Methods and compositions for treatment of bone defects with placental cell populations

ABSTRACT

Provided herein are methods of using adherent placental stem cells and placental stem cell populations, and methods of culturing, proliferating and expanding the same. Also provided herein are methods of differentiating the placental stem cells. Further provided herein are methods of using the placental stem cells to formulate implantable or injectable compositions suitable for administration to a subject. Still further provided herein are provides methods for treating bone defects with stem cells and compositions comprising stem cells.

This application is a divisional of U.S. application Ser. No.14/028,228, filed Sep. 16, 2013, which is a divisional of U.S.application Ser. No. 11/877,475, filed Oct. 23, 2007, now U.S. Pat. No.8,562,972, which claims benefit of U.S. Provisional Application No.60/853,971, filed Oct. 23, 2006; U.S. Provisional Application No.60/855,629, filed Oct. 30, 2006; and U.S. Provisional Application No.60/997,022, filed Sep. 28, 2007.

1. FIELD

Provided herein are isolated placental cells, e.g., placental perfusate,adherent and nonadherent placental stem cells, populations of placentalstem cells, compositions comprising the stem cells, methods of obtainingthe stem cells, methods of formulating compositions comprising the stemcells, and methods of treating bone defects with the stem cells andcompositions.

2. BACKGROUND

Human stem cells are totipotential or pluripotential precursor cellscapable of generating a variety of mature human cell lineages. Evidenceexists that demonstrates that stem cells can be employed to repopulatemany, if not all, tissues and restore physiologic and anatomicfunctionality.

Many different types of mammalian stem cells have been characterized.See, e.g., Caplan et al., U.S. Pat. No. 5,486,359 (human mesenchymalstem cells); Boyse et al., U.S. Pat. No. 5,004,681 (fetal and neonatalhematopoietic stem and progenitor cells); Boyse et al., U.S. Pat. No.5,192,553 (same); Beltrami et al., Cell 114(6):763-766 (2003) (cardiacstem cells); Forbes et al., J. Pathol. 197(4):510-518 (2002) (hepaticstem cells). Umbilical cord blood, and total nucleated cells derivedfrom cord blood, have been used in transplants to restore, partially orfully, hematopoietic function in patients who have undergone ablativetherapy.

3. SUMMARY

Provided herein are isolated placental cells, e.g., placental perfusate,adherent or nonadherent placental stem cells, populations of placentalstem cells, compositions comprising the cells, methods of obtaining theplacental cells, methods of formulating the compositions, and methods ofusing the cells to treat bone defects.

Provided herein are isolated stem cells, and cell populations comprisingsuch stem cells, wherein the stem cells are present in, and isolatablefrom placental tissue (e.g., amnion, chorion, placental cotyledons,umbilical cord, etc.), that are useful in the repair of bone defects.The placental stem cells exhibit one or more characteristics of a stemcell (e.g., exhibit markers associated with stem cells, replicate atleast 10-20 times in culture in an undifferentiated state, differentiateinto adult cells representative of the three germ layers, etc.), and canadhere to a tissue culture substrate (e.g., tissue culture plastic suchas the surface of a tissue culture dish or multiwell plate).

In one embodiment, provided herein is an isolated placental stem cellthat is nonadherent. In certain embodiments, the isolated stem cell isCD34⁺. In certain embodiments, the isolated stem cell is CD44⁻. Incertain embodiments, the isolated stem cell is CD34⁺ and CD44⁻. Incertain embodiments, the isolated stem cell is CD9⁺, CD54⁺, CD90⁺, orCD166⁺. In certain embodiments, the isolated stem cell is CD9⁺, CD54⁺,CD90⁺, and CD166⁺. In certain embodiments, the isolated stem cell isCD31⁺, CD117⁺, CD133⁺, or CD200⁺. In certain embodiments, the isolatedstem cell is CD31⁺, CD117⁺, CD133⁺, and CD200⁺. In certain embodiments,the isolated stem cell has been isolated from a human placenta byenzymatic digestion. In certain embodiments, the isolated stem cell hasbeen isolated from a human placenta by perfusion. In certainembodiments, the isolated stem cell facilitates formation of amineralized matrix in a population of placental cells when saidpopulation is cultured under conditions that allow the formation of amineralized matrix.

In another embodiment, provided herein is a population of isolatedplacental cells that are nonadherent. In certain embodiments, thepopulation comprises stem cells that are CD34⁺. In certain embodiments,the population comprises stem cells that are CD44⁻. In certainembodiments, the population comprises stem cells that are CD34⁺ andCD44⁻. In certain embodiments, the population comprises stem cells thatare CD9⁺, CD54⁺, CD90⁺, or CD166⁺. In certain embodiments, thepopulation comprises stem cells that are CD9⁺, CD54⁺, CD90⁺, and CD166⁺.In certain embodiments, the population comprises stem cells that areCD31⁺, CD117⁺, CD133⁺, or CD200⁺. In certain embodiments, the populationcomprises stem cells that are CD31⁺, CD117⁺, CD133⁺, and CD200⁺. Incertain embodiments, the population comprises stem cells, wherein atleast about 70% of said cells are CD34⁺ and CD44⁻ stem cells. In certainembodiments, the population comprises stem cells, wherein at least about90% of said cells are CD34⁺ and CD44⁻ stem cells. In certainembodiments, the population has been expanded. In certain embodiments,the population has been passaged at least once. In certain embodiments,the population has been passaged at least five times. In certainembodiments, the population has been passaged at least ten times. Incertain embodiments, the population has been passaged at least twentytimes. In certain embodiments, the population forms, or facilitates theformation of, a mineralized matrix in a population of placental cellswhen said population is cultured under conditions that allow theformation of a mineralized matrix.

In another aspect, provided herein is a population of isolated placentalstem cells that are CD34⁺ and CD44⁻. In certain embodiments, the stemcells are CD9⁺, CD54⁺, CD90⁺, or CD166⁺. In certain embodiments, thestem cells are CD9⁺, CD54⁺, CD90⁺, and CD166⁺. In certain embodiments,the stem cells are CD31⁺, CD117⁺, CD133⁺, or CD200⁺. In certainembodiments, the stem cells are CD31⁺, CD117⁺, CD133⁺, and CD200⁺. Incertain embodiments, at least about 70% of the stem cells are CD34⁺ andCD44⁻ stem cells. In certain embodiments, at least about 90% of the stemcells are CD34⁺ and CD44⁻ stem cells. In certain embodiments, thepopulation has been expanded. In certain embodiments, the population hasbeen passaged at least once. In certain embodiments, the population hasbeen passaged at least five times. In certain embodiments, thepopulation has been passaged at least ten times. In certain embodiments,the population has been passaged at least twenty times. In certainembodiments, the population forms, or facilitates the formation of, amineralized matrix in a population of placental cells when saidpopulation is cultured under conditions that allow the formation of amineralized matrix.

In one embodiment, provided herein is an isolated placental stem cellthat is CD200⁺ or HLA-G⁺. In a specific embodiment, the stem cell isadherent. In another specific embodiment, said cell is CD200⁺ andHLA-G⁺. In a specific embodiment, said stem cell is CD73⁺ and CD105⁺. Inanother specific embodiment, said stem cell is CD34⁻, CD38⁻ or CD45⁻. Inanother specific embodiment, said stem cell is CD34⁻, CD38⁻ and CD45⁻.In another specific embodiment, said stem cell is CD34⁻, CD38⁻, CD45⁻,CD73⁺ and CD105⁺. In another specific embodiment, said stem cellfacilitates the formation of one or more embryoid-like bodies from apopulation of isolated placental cells comprising placental stem cellswhen said population is cultured under conditions that allow formationof embryoid-like bodies.

In another embodiment, provided herein is a population of isolatedplacental cells comprising CD200⁺, HLA-G⁺ stem cells. In a specificembodiment, said stem cells are adherent. In various embodiments, atleast about 10%, at least about 20%, at least about 30%, at least about40%, at least about 50% at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, or at least about 95% or more of saidisolated placental cells are CD200⁺, HLA-G⁺ stem cells. In a specificembodiment of the above populations, said stem cells are CD73⁺ andCD105⁺. In another specific embodiment, said stem cells are CD34⁻, CD38⁻or CD45⁻. In a more specific embodiment, said stem cells are CD34⁻,CD38⁻, CD45⁻, CD73⁺ and CD105⁺. In other specific embodiments, saidpopulation has been expanded, e.g., passaged at least once, at leastthree times, at least five times, at least 10 times, at least 15 times,or at least 20 times. In another specific embodiment, said populationforms one or more embryoid-like bodies when cultured under conditionsthat allow formation of embryoid-like bodies.

In another embodiment, provided herein is an isolated placental stemcell that is CD73⁺, CD105⁺, and CD200⁺. In a specific embodiment, saidstem cell is adherent. In another specific embodiment, said stem cell isHLA-G⁺. In another specific embodiment, said stem cell is CD34⁻, CD38⁻or CD45⁻. In another specific embodiment, said stem cell is CD34⁻, CD38⁻and CD45⁻. In a more specific embodiment, said stem cell is CD34⁻,CD38⁻, CD45⁻, and HLA-G⁺. In another specific embodiment, said stem cellfacilitates development of one or more embryoid-like bodies from apopulation of isolated placental cells comprising the stem cell whensaid population is cultured under conditions that allow formation ofembryoid-like bodies.

In another embodiment, provided herein is a population of isolatedplacental cells comprising CD73⁺, CD105⁺, CD200⁺ stem cells. In aspecific embodiment, said stem cells are adherent. In variousembodiments, at least about 10%, at least about 20%, at least about 30%,at least about 40%, at least about 50% at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, or at least about 95%of said isolated placental cells are CD73⁺, CD105⁺, CD200⁺ stem cells.In a specific embodiment of said populations, said stem cells areHLA-G⁺. In another specific embodiment, said stem cells are CD34⁻, CD38⁻or CD45⁻. In another specific embodiment, said stem cells are CD34⁻,CD38⁻ and CD45⁻. In a more specific embodiment, said stem cells areCD34⁻, CD38⁻, CD45⁻, and HLA-G⁺. In other specific embodiments, saidpopulation has been expanded, for example, passaged at least once, atleast three times, at least five times, at least 10 times, at least 15times, or at least 20 times. In another specific embodiment, saidpopulation forms one or more embryoid-like bodies in culture underconditions that allow formation of embryoid-like bodies.

Also provided herein is an isolated placental stem cell that is CD200⁺and OCT-4⁺. In a specific embodiment, said stem cell is adherent. Inanother specific embodiment, the stem cell is CD73⁺ and CD105⁺. Inanother specific embodiment, said stem cell is HLA-G⁺. In anotherspecific embodiment, said stem cell is CD34⁻, CD38⁻ or CD45⁻. In anotherspecific embodiment, said stem cell is CD34⁻, CD38⁻ and CD45⁻. In a morespecific embodiment, said stem cell is CD34⁻, CD38⁻, CD45⁻, CD73⁺,CD105⁺ and HLA-G⁺. In another specific embodiment, said stem cellfacilitates the formation of one or more embryoid-like bodies from apopulation of isolated placental cells comprising placental stem cellswhen said population is cultured under conditions that allow formationof embryoid-like bodies.

In another embodiment, provided herein is a population of isolatedplacental cells comprising CD200⁺, OCT-4⁺ placental stem cells. In aspecific embodiment, the stem cells are adherent. In variousembodiments, at least about 10%, at least about 20%, at least about 30%,at least about 40%, at least about 50% at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, or at least about 95%of said isolated placental cells are CD200⁺, OCT-4⁺ stem cells. In aspecific embodiment of the above populations, said stem cells are CD73⁺and CD105⁺. In another specific embodiment, said stem cells are HLA-G⁺.In another specific embodiment, said stem cells are CD34⁻, CD38⁻ andCD45⁻. In a more specific embodiment, said stem cells are CD34⁻, CD38⁻,CD45⁻, CD73⁺, CD105⁺ and HLA-G⁺. In other specific embodiments, saidpopulation has been expanded, for example, has been passaged at leastonce, at least three times, at least five times, at least 10 times, atleast 15 times, or at least 20 times. In another specific embodiment,said population forms one or more embryoid-like bodies when culturedunder conditions that allow the formation of embryoid-like bodies.

In another embodiment, provided herein is an isolated placental stemcell that is CD73⁺ and CD105⁺ and which facilitates the formation of oneor more embryoid-like bodies in a population of isolated placental cellscomprising said stem cell when said population is cultured underconditions that allow formation of embryoid-like bodies. In a specificembodiment, said stem cell is adherent. In another specific embodiment,said stem cell is CD34⁻, CD38⁻ or CD45⁻. In another specific embodiment,said stem cell is CD34⁻, CD38⁻ and CD45⁻. In another specificembodiment, said stem cell is OCT4⁺. In a more specific embodiment, saidstem cell is OCT4+, CD34⁻, CD38⁻ and CD45⁻.

Further provided herein is a population of isolated placental cellscomprising CD73⁺, CD105⁺ placental stem cells, wherein said populationforms one or more embryoid-like bodies under conditions that allowformation of embryoid-like bodies. In a specific embodiment, said stemcells are adherent. In various embodiments, at least about 10%, at leastabout 20%, at least about 30%, at least about 40%, at least about 50% atleast about 60%, at least about 70%, at least about 80%, at least about90%, or at least about 95% of said isolated placental cells are CD73⁺,CD105⁺ stem cells. In a specific embodiment of the above populations,said stem cells are CD34⁻, CD38⁻ or CD45⁻. In another specificembodiment, said stem cells are CD34⁻, CD38⁻ and CD45⁻. In anotherspecific embodiment, said stem cells are OCT-4⁺. In a more specificembodiment, said stem cells are OCT-4⁺, CD34⁻, CD38⁻ and CD45⁻. In otherspecific embodiments, said population has been expanded, for example,has been passaged at least once, at least three times, at least fivetimes, at least 10 times, at least 15 times, or at least 20 times.

Further provided herein is an isolated placental stem cell that isCD73⁺, CD105⁺ and HLA-G⁺. In a specific embodiment, said stem cell isadherent. In another specific embodiment, said stem cell is CD34⁻, CD38⁻or CD45⁻. In another specific embodiment, said stem cell is CD34⁻, CD38⁻and CD45⁻. In another specific embodiment, said stem cell is OCT-4⁺. Inanother specific embodiment, said stem cell is CD200⁺. In a morespecific embodiment, said stem cell is CD34⁻, CD38⁻, CD45⁻, OCT-4⁺ andCD200⁺. In another specific embodiment, said stem cell facilitates theformation of one or more embryoid-like bodies from a population ofisolated placental cells comprising placental stem cells in cultureunder conditions that allow formation of embryoid-like bodies.

Further provided herein is a population of isolated placental cellscomprising CD73⁺, CD105⁺ and HLA-G⁺ placental stem cells. In a specificembodiment, the stem cells are adherent. In various embodiments, atleast about 10%, at least about 20%, at least about 30%, at least about40%, at least about 50% at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, or at least about 95% of said isolatedplacental cells are CD73⁺, CD105⁺ and HLA-G⁺ stem cells. In a specificembodiment of the above populations, said stem cells are CD34⁻, CD38⁻ orCD45⁻. In another specific embodiment, said stem cells are CD34⁻, CD38⁻and CD45⁻. In another specific embodiment, said stem cells are OCT-4⁺.In another specific embodiment, said stem cells are CD200⁺. In a morespecific embodiment, said stem cells are CD34⁻, CD38⁻, CD45⁻, OCT-4⁺ andCD200⁺. In another specific embodiment, said population has beenexpanded, for example, has been passaged at least once, at least threetimes, at least five times, at least 10 times, at least 15 times, or atleast 20 times. In another specific embodiment, said population formsembryoid-like bodies when cultured under conditions that allow theformation of embryoid-like bodies.

Further provided herein is an isolated placental stem cell that isOCT-4⁺ and which facilitates formation of one or more embryoid-likebodies in a population of isolated placental cells comprising said stemcell when cultured under conditions that allow formation ofembryoid-like bodies. In a specific embodiment, said stem cell isadherent. In another specific embodiment, said stem cell is CD73⁺ andCD105⁺. In another specific embodiment, said stem cell is CD34⁻, CD38⁻,or CD45⁻. In another specific embodiment, said stem cell is CD200⁺. In amore specific embodiment, said stem cell is CD73⁺, CD105⁺, CD200⁺,CD34⁻, CD38⁻, and CD45⁻.

Also provided herein is a population of isolated placental cellscomprising OCT-4⁺ placental stem cells, wherein said population formsone or more embryoid-like bodies when cultured under conditions thatallow the formation of embryoid-like bodies. In a specific embodiment,the stem cells are adherent. In various embodiments, at least about 10%,at least about 20%, at least about 30%, at least about 40%, at leastabout 50% at least about 60%, at least about 70%, at least about 80%, atleast about 90%, or at least about 95% of said isolated placental cellsare OCT4⁺ stem cells. In a specific embodiment of the above populations,said stem cells are CD73⁺ and CD105⁺. In another specific embodiment,said stem cells are CD34⁻, CD38⁻, or CD45⁻. In another specificembodiment, said stem cells are CD200⁺. In a more specific embodiment,said stem cells are CD73⁺, CD105⁺, CD200⁺, CD34⁻, CD38⁻, and CD45⁻. Inanother specific embodiment, said population has been expanded, forexample, passaged at least once, at least three times, at least fivetimes, at least 10 times, at least 15 times, or at least 20 times.

Further provided herein is an isolated population of the adherent ornonadherent placental stem cells described herein that is producedaccording to a method comprising perfusing a mammalian placenta that hasbeen drained of cord blood and perfused to remove residual blood;perfusing said placenta with a perfusion solution; and collecting saidperfusion solution, wherein said perfusion solution after perfusioncomprises a population of placental cells that comprises placental stemcells; and isolating a plurality of said placental stem cells from saidpopulation of cells. In a specific embodiment, the perfusion solution ispassed through both the umbilical vein and umbilical arteries andcollected after it exudes from the placenta. In another specificembodiment, the perfusion solution is passed through the umbilical veinand collected from the umbilical arteries, or passed through theumbilical arteries and collected from the umbilical vein.

Further provided herein is an isolated placental stem cell, or isolatedpopulation of the placental stem cells, described herein that isproduced according to a method comprising digesting placental tissuewith a tissue-disrupting enzyme to obtain a population of placentalcells comprising placental stem cells, and isolating a plurality ofplacental stem cells from the remainder of said placental cells. Inspecific embodiments, said placental tissue is a whole placenta, anamniotic membrane, chorion, a combination of amnion and chorion, or acombination of any of the foregoing. In other specific embodiment, thetissue-disrupting enzyme is trypsin or collagenase.

In more specific embodiments, provided herein is an isolated placentalstem cell, wherein said stem cell expresses one or more genes at adetectably higher level than a bone marrow-derived mesenchymal stemcell, wherein said one or more genes are ACTG2, ADARB1, AMIGO2, ATRS-1,B4GALT6, BCHE, C11orf9, CD200, COL4A1, COL4A2, CPA4, DMD, DSC3, DSG2,ELOVL2, F2RL1, FLJ10781, GATA6, GPR126, GPRC5B, ICAM1, IER3, IGFBP7,IL1A, IL6, IL18, KRT18, KRT8, LIPG, LRAP, MATN2, MEST, NFE2L3, NUAK1,PCDH7, PDLIM3, PJP2, RTN1, SERPINB9, ST3GAL6, ST6GALNAC5, SLC12A8,TCF21, TGFB2, VTN, and/or ZC3H12A, and wherein said bone marrow derivedstem cell has undergone a number of passages in culture equivalent to anumber of passages for said placental stem cell. In a more specificembodiment, said placental stem cell expresses ACTG2, ADARB1, AMIGO2,ATRS-1, B4GALT6, BCHE, C11orf9, CD200, COL4A1, COL4A2, CPA4, DMD, DSC3,DSG2, ELOVL2, F2RL1, FLJ10781, GATA6, GPR126, GPRC5B, ICAM1, IER3,IGFBP7, IL1A, IL6, IL18, KRT18, KRT8, LIPG, LRAP, MATN2, MEST, NFE2L3,NUAK1, PCDH7, PDLIM3, PJP2, RTN1, SERPINB9, ST3GAL6, ST6GALNAC5,SLC12A8, TCF21, TGFB2, VTN, and ZC3H12A at a detectably higher levelthan a bone marrow-derived mesenchymal stem cell.

In more specific embodiments, also provided herein is a population ofisolated placental stem cells, wherein said population of stem cellsexpress one or more genes at a detectably higher level than a populationof bone marrow-derived mesenchymal stem cells, wherein said one or moregenes are ACTG2, ADARB1, AMIGO2, ATRS-1, B4GALT6, BCHE, C11orf9, CD200,COL4A1, COL4A2, CPA4, DMD, DSC3, DSG2, ELOVL2, F2RL1, FLJ10781, GATA6,GPR126, GPRC5B, ICAM1, IER3, IGFBP7, IL1A, IL6, IL18, KRT18, KRT8, LIPG,LRAP, MATN2, MEST, NFE2L3, NUAK1, PCDH7, PDLIM3, PJP2, RTN1, SERPINB9,ST3GAL6, ST6GALNAC5, SLC12A8, TCF21, TGFB2, VTN, and/or ZC3H12A, andwherein said population of bone marrow derived stem cells has undergonea number of passages in culture equivalent to a number of passages forsaid placental stem cell, and wherein said population of bonemarrow-derived mesenchymal stem cells has a number of cells equivalentto said population of isolated stem cells. In a more specificembodiment, the population of isolated stem cells expresses ACTG2,ADARB1, AMIGO2, ATRS-1, B4GALT6, BCHE, C11orf9, CD200, COL4A1, COL4A2,CPA4, DMD, DSC3, DSG2, ELOVL2, F2RL1, FLJ10781, GATA6, GPR126, GPRC5B,ICAM1, IER3, IGFBP7, IL1A, IL6, IL18, KRT18, KRT8, LIPG, LRAP, MATN2,MEST, NFE2L3, NUAK1, PCDH7, PDLIM3, PJP2, RTN1, SERPINB9, ST3GAL6,ST6GALNAC5, SLC12A8, TCF21, TGFB2, VTN, and ZC3H12A at a detectablyhigher level than said population of isolated bone marrow-derivedmesenchymal stem cells.

Also provided herein are compositions that comprise one or more of theplacental cells, e.g., placental perfusate, placental perfusate cells orplacental stem cells, provided herein, wherein the cells have beenisolated from the placenta. In preferred embodiments, the compositionscomprising placental cells are useful for the repair of bone defects.Thus, provided herein is a composition comprising placental perfusate,or cells isolated from placental perfusate, e.g., total nucleated cellsfrom placental perfusate.

In one aspect, provided herein is a composition comprising placentalperfusate or placental perfusate cells, e.g., total nucleated cells fromplacental perfusate.

Further provided herein is a composition comprising a placental stemcell, wherein said stem cell is an isolated placental stem cell that isnonadherent. In certain embodiments, the stem cell is CD34⁺. In certainembodiments, the stem cell is CD44⁻. In certain embodiments, the stemcell is CD34⁺ and CD44⁻. In certain embodiments, the stem cell is CD9⁺,CD54⁺, CD90⁺, or CD166⁺. In certain embodiments, the stem cell is CD9⁺,CD54⁺, CD90⁺, and CD166⁺. In certain embodiments, the stem cell isCD31⁺, CD117⁺, CD133⁺, or CD200⁺. In certain embodiments, the stem cellis CD31⁺, CD117⁺, CD133⁺, and CD200⁺. In certain embodiments, the stemcell has been isolated from a human placenta by enzymatic digestion. Incertain embodiments, the stem cell has been isolated from a humanplacenta by perfusion. In certain embodiments, the cell facilitatesformation of a mineralized matrix in a population of placental cellswhen said population is cultured under conditions that allow theformation of a mineralized matrix.

In another aspect, provided herein is a composition comprising aplacental stem cell, wherein said stem cell is an isolated stem cellthat is CD34⁺ and CD44⁻. In certain embodiments, the stem cell is CD9⁺,CD54⁺, CD90⁺, or CD166⁺. In certain embodiments, the stem cell is CD9⁺,CD54⁺, CD90⁺, and CD166⁺. In certain embodiments, the stem cell isCD31⁺, CD117⁺, CD133⁺, or CD200⁺. In certain embodiments, the stem cellis CD31⁺, CD117⁺, CD133⁺, and CD200⁺. In certain embodiments, the stemcell has been isolated from a human placenta by enzymatic digestion. Incertain embodiments, the stem cell has been isolated from a humanplacenta by perfusion. In certain embodiments, the cell facilitatesformation of a mineralized matrix in a population of placental cellswhen said population is cultured under conditions that allow theformation of a mineralized matrix.

In certain embodiments, the composition comprises an isolated stem cellprovided herein and a compound that induces the differentiation of saidstem cell into an osteogenic cell. In certain embodiments, thecomposition comprises an isolated stem cell, or a population of isolatedstem cells, provided herein, and a compound that induces thedifferentiation of a plurality of stem cells in said population of stemcells into osteogenic cells. In certain embodiments, the compound isdexamethasone or ascorbic acid.

In certain embodiments, provided herein is a composition comprising anisolated placental stem cell, wherein said stem cell is CD200⁺ andHLA-G⁺. In a specific embodiment, the stem cell is adherent. In anotherspecific embodiment, said stem cell is CD73⁺ and CD105⁺. In anotherspecific embodiment, said stem cell is CD34⁻, CD38⁻ or CD45⁻. In anotherspecific embodiment, said stem cell is CD34⁻, CD38⁻ and CD45⁻. In a morespecific embodiment, said stem cell is CD34⁻, CD38⁻, CD45⁻, CD73⁺,CD105⁺, CD200⁺ and HLA-G⁺.

In another embodiment, provided herein is a composition comprising anisolated placental stem cell, wherein said stem cell is CD73⁺, CD105⁺and CD200⁺. In a specific embodiment, the stem cell is adherent. Inanother specific embodiment, said stem cell is HLA-G⁺. In anotherspecific embodiment, said stem cell is CD34⁻, CD38⁻ or CD45⁻. In anotherspecific embodiment, said stem cell is CD34⁻, CD38⁻ and CD45⁻. Inanother specific embodiment, said stem cell is CD34⁻, CD38⁻, CD45⁻, andHLA-G⁺.

In another embodiment, provided herein is a composition comprising anisolated placental stem cell, wherein said stem cell is CD200⁺ andOCT-4⁺. In a specific embodiment, the stem cell is adherent. In anotherspecific embodiment, said stem cell is CD73⁺ and CD105⁺. In anotherspecific embodiment, said stem cell is HLA-G⁺. In another specificembodiment, said stem cell is CD34⁻, CD38⁻ or CD45⁻. In another specificembodiment, said stem cell is CD34⁻, CD38⁻ and CD45⁻. In anotherspecific embodiment, said stem cell is CD34⁻, CD38⁻, CD45⁻, CD73⁺,CD105⁺, and HLA-G⁺.

In another embodiment, provided herein is a composition comprising anisolated placental stem cell that is CD73⁺ and CD105⁺, wherein said stemcell facilitates formation of an embryoid-like body in a population ofisolated placental cells comprising said stem cell under conditions thatallow the formation of an embryoid-like body. In a specific embodiment,the stem cell is adherent. In another specific embodiment, said stemcell is CD34⁻, CD38⁻ or CD45⁻. In another specific embodiment, said stemcell is OCT-4⁺. In another specific embodiment, said stem cell isCD200⁺. In another specific embodiment, said stem cell is OCT-4+,CD200⁺, CD34⁻, CD38⁻ and CD45⁻.

In yet another embodiment, provided herein is a composition comprisingan isolated placental stem cell that is CD73⁺, CD105⁺ and HLA-G⁺. In aspecific embodiment, the stem cell is adherent. In another specificembodiment, said stem cell is CD34⁻, CD38⁻ or CD45⁻. In another specificembodiment, said stem cell is OCT-4⁺. In another specific embodiment,said stem cell is CD200⁺. In another specific embodiment, said stem cellis OCT-4+, CD200⁺, CD34⁻, CD38⁻ and CD45.

In another embodiment, provided herein is a composition comprising anisolated placental stem cell that is OCT-4⁺, wherein said stem cellfacilitates formation of an embryoid-like body in a population ofisolated placental cells comprising said stem cell under conditions thatallow the formation of an embryoid-like body. In a specific embodiment,said stem cell is CD73⁺ and CD105⁺. In another specific embodiment, saidstem cell is CD34⁻, CD38⁻ and CD45⁻. In another specific embodiment,said stem cell is CD200⁺. In another specific embodiment, said stem cellis CD73⁺, CD105⁺, CD200⁺, CD34⁻, CD38⁻ and CD45⁻.

Further provided herein is a composition comprising a placental stemcells that expresses one or more genes at a detectably higher level thana bone marrow-derived mesenchymal stem cell, wherein said one or moregenes are selected from the group consisting of ACTG2, ADARB1, AMIGO2,ATRS-1, B4GALT6, BCHE, C11orf9, CD200, COL4A1, COL4A2, CPA4, DMD, DSC3,DSG2, ELOVL2, F2RL1, FLJ10781, GATA6, GPR126, GPRC5B, ICAM1, IER3,IGFBP7, IL1A, IL6, IL18, KRT18, KRT8, LIPG, LRAP, MATN2, MEST, NFE2L3,NUAK1, PCDH7, PDLIM3, PJP2, RTN1, SERPINB9, NAI-1502268926v1 ST3GAL6,ST6GALNAC5, SLC12A8, TCF21, TGFB2, VTN, and ZC3H12A, and wherein saidbone marrow derived stem cell has undergone a number of passages inculture equivalent to a number of passages for said placental stem cell.In a more specific embodiment of the above composition, said stem cellsexpress ACTG2, ADARB1, AMIGO2, ATRS-1, B4GALT6, BCHE, C11orf9, CD200,COL4A1, COL4A2, CPA4, DMD, DSC3, DSG2, ELOVL2, F2RL1, FLJ10781, GATA6,GPR126, GPRC5B, ICAM1, IER3, IGFBP7, IL1A, IL6, IL18, KRT18, KRT8, LIPG,LRAP, MATN2, MEST, NFE2L3, NUAK1, PCDH7, PDLIM3, PJP2, RTN1, SERPINB9,ST3GAL6, ST6GALNAC5, SLC12A8, TCF21, TGFB2, VTN, and ZC3H12A at adetectably higher level than a population of isolated bonemarrow-derived mesenchymal stem cell, wherein said population of stemcells and said population of bone marrow-derived mesenchymal cells haveequivalent numbers of cells.

In another specific embodiment, any of the foregoing compositionscomprises a matrix. In a more specific embodiment, said matrix is athree-dimensional scaffold. In another more specific embodiment, saidmatrix comprises collagen, gelatin, laminin, fibronectin, pectin,ornithine, or vitronectin. In another more specific embodiment, thematrix is an amniotic membrane or an amniotic membrane-derivedbiomaterial. In another more specific embodiment, said matrix comprisesan extracellular membrane protein. In another more specific embodiment,said matrix comprises a synthetic compound. In another more specificembodiment, said matrix comprises a bioactive compound. In another morespecific embodiment, said bioactive compound is a growth factor,cytokine, antibody, or organic molecule of less than 5,000 daltons. Incertain embodiments, the matrix is a synthetic degradable polymer suchas, for example, polylactic acid or polyglycolic acid. In certainembodiments, the matrix is an implantable scaffolding substrate. Incertain embodiments, the implantable scaffolding substrate is selectedfrom the group consisting of a β-tricalcium phosphate substrate, aβ-tricalcium phosphate-collagen substrate, a collagen substrate, acalcium phosphate substrate, a mineralized human placental collagensubstrate, a hyaluronic acid substrate, and a ceramic substrate. Incertain embodiments, the implantable scaffolding substrate is aβ-tricalcium phosphate substrate. In certain embodiments, theimplantable scaffolding substrate is a β-tricalcium phosphate-collagensubstrate. In certain embodiments, the implantable scaffolding substrateis a collagen substrate. In certain embodiments, the implantablescaffolding substrate is a calcium phosphate substrate. In certainembodiments, the implantable scaffolding substrate is a mineralizedhuman placental collagen substrate.

In another embodiment, further provided herein is a compositioncomprising medium conditioned by any of the foregoing stem cells, or anyof the foregoing stem cell populations. In a specific embodiment, anysuch composition comprises a stem cell that is not derived from aplacenta. In a more specific embodiment, said stem cell is an embryonicstem cell. In another more specific embodiment, said stem cell is amesenchymal stem cell. In another more specific embodiment, said stemcell is a bone marrow-derived stem cell. In another more specificembodiment, said stem cell is a hematopoietic progenitor cell. Inanother more specific embodiment, said stem cell is a somatic stem cell.In an even more specific embodiment, said somatic stem cell is a neuralstem cell, a hepatic stem cell, a pancreatic stem cell, an endothelialstem cell, a cardiac stem cell, or a muscle stem cell.

In another aspect, provided herein is a composition comprising mediumconditioned by a placental stem cell or population of placental stemcells provided herein. In certain embodiments, the composition comprisesmedium conditioned by a cell population, e.g., a stem cell population,provided herein.

Also provided herein is a method of producing a cell populationcomprising selecting cells that do not adhere to a substrate, andisolating said cells from other cells to form a cell population. Incertain embodiments, the method further comprises selecting cells thatexpress CD34 and do not express CD44 and increasing the concentrationof, e.g., isolating said cells from other cells, to form a cellpopulation.

In certain embodiments, provided herein is a method of producing a cellpopulation, comprising selecting cells that (a) do not adhere to asubstrate, (b) express CD34 and do not express CD44, and (c) facilitatethe formation of mineralized matrix in a population of placental cellswhen said population is cultured under conditions that allow for theformation of a mineralized matrix; and isolating said cells from othercells to form a cell population. In certain embodiments, the substratecomprises fibronectin.

In certain embodiments, the method further comprises selecting cellsthat express CD9, CD29, CD54, CD90, CD166, or a combination of theforegoing.

In certain embodiments, the method further comprises selecting cellsthat express CD31, CD34, CD117, CD133, CD200, or a combination of theforegoing.

In certain embodiments, the selecting is accomplished using an antibody.In certain embodiments, the selecting is accomplished using flowcytometry. In certain embodiments, the selecting is accomplished usingmagnetic beads. In certain embodiments, the selecting is accomplished byfluorescence-activated cell sorting. In certain embodiments, the cellpopulation is expanded.

In another aspect, provided herein is a population of nonadherentplacental stem cells, wherein said cells have been cryopreserved, andwherein said population is contained within a container. In certainembodiments, the stem cells are CD34⁺ and CD44⁻. In certain embodiments,the cells have been cryopreserved, and wherein said population iscontained within a container, and wherein said stem cells form amineralized matrix when cultured under conditions allowing the formationof a mineralized matrix. In certain embodiments, the container is a bagsuitable for the intravenous delivery of a liquid. In certainembodiments, the population comprises 1×10⁶ said stem cells. In certainembodiments, the population comprises 5×10⁶ said stem cells. In certainembodiments, the population comprises 1×10⁷ said stem cells. In certainembodiments, the population comprises 5×10⁷ said stem cells. In certainembodiments, the population comprises 1×10⁸ said stem cells. In certainembodiments, the population comprises 5×10⁸ said stem cells. In certainembodiments, the population comprises 1×10⁹ said stem cells. In certainembodiments, the population comprises 5×10⁹ said stem cells. In certainembodiments, the population comprises 1×10¹⁰ said stem cells. In certainembodiments, the stem cells have been passaged no more than 5 times. Incertain embodiments, the stem cells have been passaged no more than 10times. In certain embodiments, the stem cells have been passaged no morethan 15 times. In certain embodiments, the stem cells have been passagedno more than 20 times. In certain embodiments, the stem cells have beenexpanded within said container. In certain embodiments, the populationis contained in a 0.9% NaCl solution.

In another aspect, provided herein is a method of producing osteogeniccells with the ability to mineralize matrix, comprising culturing aplurality of stem cells provided herein or a population of isolated stemcells provided herein, under conditions in which said stem cellsdifferentiate into osteogenic cells, said culturing being for a timesufficient for said osteogenic cells to produce, or facilitate theproduction of, detectable amounts of mineralized matrix rich in calciumand/or phosphate. In certain embodiments, the osteogenic cells producebone.

In still another aspect, provided herein is a method for formulating amatrix, comprising combining a population of stem cells provided hereinwith an implantable scaffolding substrate. In certain embodiments, thestem cells are nonadherent. In certain embodiments, the stem cells areCD34⁺. In certain embodiments, the stem cells are CD44⁻. In certainembodiments, the stem cells are CD34⁺ and CD44⁻. In certain embodiments,the stem cells are CD9⁺, CD54⁺, CD90⁺, or CD166⁺. In certainembodiments, the stem cells are CD9⁺, CD54⁺, CD90⁺, and CD166⁺. Incertain embodiments, the stem cells are CD31⁺, CD117⁺, CD133⁺, orCD200⁺. In certain embodiments, the stem cells are CD31⁺, CD117⁺,CD133⁺, and CD200⁺. In certain embodiments, at least about 70% of thestem cells are CD34⁺ and CD44⁻ stem cells. In certain embodiments, atleast about 90% of the stem cells are CD34⁺ and CD44⁻ stem cells. Incertain embodiments, the population comprises 1×10⁶ said stem cells. Incertain embodiments, the population comprises 5×10⁶ said stem cells. Incertain embodiments, the population comprises 1×10⁷ said stem cells. Incertain embodiments, the population comprises 5×10⁷ said stem cells. Incertain embodiments, the population comprises 1×10⁸ said stem cells. Incertain embodiments, the population comprises 5×10⁸ said stem cells. Incertain embodiments, the population comprises 1×10⁹ said stem cells. Incertain embodiments, the population comprises 5×10⁹ said stem cells. Incertain embodiments, the population comprises 1×10¹⁰ said stem cells. Incertain embodiments, the stem cells have been passaged at least, about,or no more than 5 times. In certain embodiments, the stem cells havebeen passaged at least, about, or no more than 10 times. In certainembodiments, the stem cells have been passaged at least, about, or nomore than 15 times. In certain embodiments, the stem cells have beenpassaged at least, about, or no more than 20 times. In certainembodiments, the population has been expanded.

In certain embodiments, the implantable scaffolding substrate isselected from the group consisting of a β-tricalcium phosphatesubstrate, a β-tricalcium phosphate-collagen substrate, a collagensubstrate, a calcium phosphate substrate, a mineralized human placentalcollagen substrate, a hyaluronic acid substrate, and a ceramicsubstrate. In certain embodiments, the implantable scaffolding substrateis a β-tricalcium phosphate substrate. In certain embodiments, theimplantable scaffolding substrate is a β-tricalcium phosphate-collagensubstrate. In certain embodiments, the implantable scaffolding substrateis a collagen substrate. In certain embodiments, the implantablescaffolding substrate is a calcium phosphate substrate. In certainembodiments, the implantable scaffolding substrate is a mineralizedhuman placental collagen substrate.

In another aspect, provided herein is a method for formulating aninjectable composition, comprising combining a population of placentalstem cells with injectable hyaluronic acid or collagen. In certainembodiments, the stem cells are nonadherent. In certain embodiments, thestem cells are CD34⁺. In certain embodiments, the stem cells are CD44⁻.In certain embodiments, the said stem cells are CD34⁺ and CD44⁻. Incertain embodiments, the said stem cells are CD9⁺, CD54⁺, CD90⁺, orCD166⁺. In certain embodiments, the said stem cells are CD9⁺, CD54⁺,CD90⁺, and CD166⁺. In certain embodiments, the said stem cells areCD31⁺, CD117⁺, CD133⁺, or CD200⁺. In certain embodiments, the said stemcells are CD31⁺, CD117⁺, CD133⁺, and CD200⁺. In certain embodiments, atleast about 70% of said cells are CD34⁺ and CD44⁻ stem cells. In certainembodiments, the at least about 90% of said cells are CD34⁺ and CD44⁻stem cells. In certain other embodiments, the placental stem cells areadherent. In specific embodiments, the placental stem cells are CD200⁺and HLA-G⁺; CD73⁺, CD105⁺, and CD200⁺; CD200⁺ and OCT-4⁺; CD73⁺, CD105⁺and HLA-G⁺; CD73⁺ and CD105⁺ and facilitates the formation of one ormore embryoid-like bodies in a population of placental cells comprisingsaid stem cell when said population is cultured under conditions thatallow the formation of an embryoid-like body; or OCT-4⁺ and facilitatesthe formation of one or more embryoid-like bodies in a population ofplacental cells comprising the stem cell when said population iscultured under conditions that allow formation of embryoid-like bodies;or any combination thereof. In more specific embodiments of thenonadherent placental stem cells, the isolated CD200⁺, HLA-G⁺ stem cellis CD34⁻, CD38⁻, CD45⁻, CD73⁺ and CD105⁺; the isolated CD73⁺, CD105⁺,and CD200⁺ stem cell is CD34⁻, CD38⁻, CD45⁻, and HLA-G⁺; the isolatedCD200⁺, OCT-4⁺ stem cell is CD34⁻, CD38⁻, CD45⁻, CD73⁺, CD105⁺ andHLA-G⁺; the isolated stem cell of claim 1, wherein said CD73⁺, CD105⁺and HLA-G⁺ stem cell is CD34⁻, CD45⁻, OCT-4⁺ and CD200⁺; the isolatedCD73⁺ and CD105⁺ stem cell that facilitates the formation of one or moreembryoid-like bodies is OCT4⁺, CD34⁻, CD38⁻ and CD45⁻; and/or theisolated OCT-4⁺ and which facilitates the formation of one or moreembryoid-like bodies is CD73⁺, CD105⁺, CD200⁺, CD34⁻, CD38⁻, and CD45⁻.In certain embodiments, the population of placental stem cells has beenexpanded. In certain embodiments, the said composition comprisesinjectable hyaluronic acid. In certain embodiments, the compositioncomprises injectable collagen. Provided herein are also compositionscomprising a population of nonadherent stem cells and injectablehyaluronic acid or collagen.

In another aspect, provided herein is a method for treating bone defectsin a subject, comprising administering to a subject in need thereof animplantable or injectable composition comprising a population of stemcells provided herein, thereby treating the bone defect in the subject.In certain embodiments, the bone defect is an osteolytic lesionassociated with a cancer, a bone fracture, or a spine, e.g., in need offusion. In certain embodiments, the osteolytic lesion is associated withmultiple myeloma, bone cancer, or metastatic cancer. In certainembodiments, the bone fracture is a non-union fracture. In certainembodiments, an implantable composition comprising a population ofnonadherent stem cells is administered to the subject. In certainembodiments, an implantable composition is surgically implanted, e.g.,at the site of the bone defect. In certain embodiments, an injectablecomposition comprising a population of nonadherent stem cells isadministered to the subject. In certain embodiments, an injectablecomposition is surgically administered to the region of the bone defect.In certain embodiments, the injectable composition is systemicallyadministered.

In certain embodiments, the stem cells are nonadherent. In certainembodiments, the stem cells are CD34⁺. In certain embodiments, the stemcells are CD44⁻. In certain embodiments, the stem cells are CD34⁺ andCD44⁻. In certain embodiments, the stem cells are CD9⁺, CD54⁺, CD90⁺, orCD166⁺. In certain embodiments, the stem cells are CD9⁺, CD54⁺, CD90⁺,and CD166⁺. In certain embodiments, the stem cells are CD31⁺, CD117⁺,CD133⁺, or CD200⁺. In certain embodiments, the stem cells are CD31⁺,CD117⁺, CD133⁺, and CD200⁺. In certain embodiments, at least about 70%of the cells are CD34⁺ and CD44⁻ stem cells. In certain embodiments, atleast about 90% of the cells are CD34⁺ and CD44⁻ stem cells. In certainother embodiments, the placental stem cells are adherent. In specificembodiments, the placental stem cells are CD200⁺ and HLA-G⁺; CD73⁺,CD105⁺, and CD200⁺; CD200⁺ and OCT-4⁺; CD73⁺, CD105⁺ and HLA-G⁺; CD73⁺and CD105⁺ and facilitates the formation of one or more embryoid-likebodies in a population of placental cells comprising said stem cell whensaid population is cultured under conditions that allow the formation ofan embryoid-like body; or OCT-4⁺ and facilitates the formation of one ormore embryoid-like bodies in a population of placental cells comprisingthe stem cell when said population is cultured under conditions thatallow formation of embryoid-like bodies; or any combination thereof. Inmore specific embodiments of the nonadherent placental stem cells, theisolated CD200⁺, HLA-G⁺ stem cell is CD34⁻, CD38⁻, CD45⁻, CD73⁺ andCD105⁺; the isolated CD73⁺, CD105⁺, and CD200⁺ stem cell is CD34⁻,CD38⁻, CD45⁻, and HLA-G⁺; the isolated CD200⁺, OCT-4⁺ stem cell isCD34⁻, CD38⁻, CD45⁻, CD73⁺, CD105⁺ and HLA-G⁺; the isolated stem cell ofclaim 1, wherein said CD73⁺, CD105⁺ and HLA-G⁺ stem cell is CD34⁻,CD45⁻, OCT-4⁺ and CD200⁺; the isolated CD73⁺ and CD105⁺ stem cell thatfacilitates the formation of one or more embryoid-like bodies is OCT4⁺,CD34⁻, CD38⁻ and CD45⁻; and/or the isolated OCT-4⁺ and which facilitatesthe formation of one or more embryoid-like bodies is CD73⁺, CD105⁺,CD200⁺, CD34⁻, CD38⁻, and CD45⁻. In certain embodiments, the populationhas been expanded.

In yet another aspect, provided herein is a method of producing a cellpopulation comprising selecting cells that a) adhere to a substrate, andb) express CD34 and do not express CD44, and isolating said cells fromother cells to form a cell population. In certain embodiments, themethod further comprises isolating said cells from other cells to form acell population. In certain embodiments, the method of producing a cellpopulation, comprises selecting cells that (a) adhere to a substrate,(b) express CD34 and do not express CD44, and (c) facilitate theformation of mineralized matrix in a population of placental cells whensaid population is cultured under conditions that allow for theformation of a mineralized matrix; and isolating said cells from othercells to form a cell population. In certain embodiments, the saidsubstrate comprises fibronectin. In certain embodiments, provided hereinis a method of producing a cell population comprising selecting cellsthat a) do not adhere to a substrate, and b) express CD34 and do notexpress CD44, and isolating said cells from other cells to form a cellpopulation. In certain embodiments, the method further comprisesisolating said cells from other cells to form a cell population. Incertain embodiments, the method of producing a cell population,comprises selecting cells that (a) do not adhere to a substrate, (b)express CD34 and do not express CD44, and (c) facilitate the formationof mineralized matrix in a population of placental cells when saidpopulation is cultured under conditions that allow for the formation ofa mineralized matrix; and isolating said cells from other cells to forma cell population. In certain embodiments, the said substrate comprisesfibronectin. In certain embodiments, the method comprises selectingcells that express at least one of the following: CD9, CD29, CD54, CD90,CD166, or a combination of the foregoing. In certain embodiments, themethod comprises selecting cells that express at least one of thefollowing: CD31, CD34, CD117, CD133, CD200, or a combination of theforegoing.

In certain embodiments, the selecting is accomplished using an antibody.In certain embodiments, the selecting is accomplished using flowcytometry. In certain embodiments, the selecting is accomplished usingmagnetic beads. In certain embodiments, the selecting is accomplished byfluorescence-activated cell sorting. In certain embodiments, the cellpopulation is expanded.

In certain embodiments, the stem cells are CD34⁺ and CD44⁻, wherein thecells have been cryopreserved, and wherein the population is containedwithin a container. In certain embodiments, the cells have beencryopreserved, and wherein said population is contained within acontainer, and wherein said stem cells form a mineralized matrix whencultured under conditions allowing the formation of a mineralizedmatrix.

In certain embodiments, the container is a bag suitable for theintravenous delivery of a liquid. In certain embodiments, the populationcomprises 1×10⁶ said stem cells. In certain embodiments, the populationcomprises 5×10⁶ said stem cells. In certain embodiments, the populationcomprises 1×10⁷ said stem cells. In certain embodiments, the populationcomprises 5×10⁷ said stem cells. In certain embodiments, the populationcomprises 1×10⁸ said stem cells. In certain embodiments, the populationcomprises 5×10⁸ said stem cells. In certain embodiments, the populationcomprises 1×10⁹ said stem cells. In certain embodiments, the comprises5×10⁹ said stem cells. In certain embodiments, the population comprises1×10¹⁰ said stem cells. In certain embodiments, the stem cells have beenpassaged no more than 5 times. In certain embodiments, the stem cellshave been passaged no more than 10 times. In certain embodiments, thestem cells have been passaged no more than 15 times. In certainembodiments, the stem cells have been passaged no more than 20 times. Incertain embodiments, the stem cells have been expanded within saidcontainer. In certain embodiments, the said population is contained in a0.9% NaCl solution.

In another aspect, provided herein is a method of producing osteogeniccells comprising culturing a plurality of placental stem cells or apopulation of isolated placental stem cells, under conditions in whichsaid stem cells differentiate into osteogenic cells, said culturingbeing for a time sufficient for said osteogenic cells to produce, orfacilitate the production of, detectable amounts of mineralized calcium.

In another aspect, provided herein is a method for formulating anmatrix, comprising combining a population of placental stem cells withan implantable scaffolding substrate, wherein said stem cells are CD34⁺and CD44⁻. In certain embodiments, the stem cells are CD9⁺, CD54⁺,CD90⁺, or CD166⁺. In certain embodiments, the stem cells are CD9⁺,CD54⁺, CD90⁺, and CD166⁺. In certain embodiments, the stem cells areCD31⁺, CD117⁺, CD133⁺, or CD200⁺. In certain embodiments, the stem cellsare CD31⁺, CD117⁺, CD133⁺, and CD200⁺. In certain embodiments, at leastabout 70% of said cells are CD34⁺ and CD44⁻ stem cells. In certainembodiments, at least about 90% of said cells are CD34⁺ and CD44⁻ stemcells. In certain embodiments, the stem cells are adherent. In specificembodiments, the adherent placental stem cells are CD200⁺ and HLA-G⁺;CD73⁺, CD105⁺, and CD200⁺; CD200⁺ and OCT-4⁺; CD73⁺, CD105⁺ and HLA-G⁺;CD73⁺ and CD105⁺ and facilitates the formation of one or moreembryoid-like bodies in a population of placental cells comprising saidstem cell when said population is cultured under conditions that allowthe formation of an embryoid-like body; or OCT-4⁺ and facilitates theformation of one or more embryoid-like bodies in a population ofplacental cells comprising the stem cell when said population iscultured under conditions that allow formation of embryoid-like bodies;or any combination thereof. In more specific embodiments of thenonadherent placental stem cells, the isolated CD200⁺, HLA-G⁺ stem cellis CD34⁻, CD38⁻, CD45⁻, CD73⁺ and CD105⁺; the isolated CD73⁺, CD105⁺,and CD200⁺ stem cell is CD34⁻, CD38⁻, CD45⁻, and HLA-G⁺; the isolatedCD200⁺, OCT-4⁺ stem cell is CD34⁻, CD38⁻, CD45⁻, CD73⁺, CD105⁺ andHLA-G⁺; the isolated stem cell of claim 1, wherein said CD73⁺, CD105⁺and HLA-G⁺ stem cell is CD34⁻, CD45⁻, OCT-4⁺ and CD200⁺; the isolatedCD73⁺ and CD105⁺ stem cell that facilitates the formation of one or moreembryoid-like bodies is OCT4⁺, CD34⁻, CD38⁻ and CD45⁻; and/or theisolated OCT-4⁺ and which facilitates the formation of one or moreembryoid-like bodies is CD73⁺, CD105⁺, CD200⁺, CD34⁻, CD38⁻, and CD45⁻.In certain embodiments, the population comprises 1×10⁶ said stem cells.In certain embodiments, the population comprises 5×10⁶ said stem cells.In certain embodiments, the population comprises 1×10⁷ said stem cells.In certain embodiments, the population comprises 5×10⁷ said stem cells.In certain embodiments, the population comprises 1×10⁸ said stem cells.In certain embodiments, the population comprises 5×10⁸ said stem cells.In certain embodiments, the population comprises 1×10⁹ said stem cells.In certain embodiments, the population comprises 5×10⁹ said stem cells.In certain embodiments, the population comprises 1×10¹⁰ said stem cells.In certain embodiments, the stem cells have been passaged no more than 5times. In certain embodiments, the stem cells have been passaged no morethan 10 times. In certain embodiments, the stem cells have been passagedno more than 15 times. In certain embodiments, the stem cells have beenpassaged no more than 20 times. In certain embodiments, the populationhas been expanded.

In certain embodiments, the implantable scaffolding substrate isselected from the group consisting of a β-tricalcium phosphatesubstrate, a β-tricalcium phosphate-collagen substrate, a collagensubstrate, a calcium phosphate substrate, a mineralized human placentalcollagen substrate, and a hyaluronic acid substrate. In certainembodiments, the implantable scaffolding substrate is a β-tricalciumphosphate substrate. In certain embodiments, the implantable scaffoldingsubstrate is a β-tricalcium phosphate-collagen substrate. In certainembodiments, the implantable scaffolding substrate is a collagensubstrate. In certain embodiments, the implantable scaffolding substrateis a calcium phosphate substrate. In certain embodiments, theimplantable scaffolding substrate is a mineralized human placentalcollagen substrate and/or scaffold.

In certain embodiments, provided herein is a method for formulating aninjectable composition, comprising combining a population of placentalstem cells with injectable hyaluronic acid or collagen, wherein saidstem cells are CD34⁺ and CD44⁻. In certain embodiments, the stem cellsare CD9⁺, CD54⁺, CD90⁺, or CD166⁺. In certain embodiments, the stemcells are CD9⁺, CD54⁺, CD90⁺, and CD166⁺. In certain embodiments, thestem cells are CD31⁺, CD117⁺, CD133⁺, or CD200⁺. In certain embodiments,the stem cells are CD31⁺, CD117⁺, CD133⁺, and CD200⁺. In certainembodiments, at least about 70% of said cells are CD34⁺ and CD44⁻ stemcells. In certain embodiments, at least about 90% of said cells areCD34⁺ and CD44⁻ stem cells. In certain embodiments, the population hasbeen expanded. In certain embodiments, the stem cells are adherent. Incertain embodiments, the composition comprises injectable hyaluronicacid. In certain embodiments, the composition comprises injectablecollagen. Also provided herein are compositions comprising a populationof nonadherent stem cells and injectable hyaluronic acid or collagen.

In yet another aspect, provided herein is a method for treating bonedefects in a subject, comprising administering to a subject in needthereof an implantable or injectable composition comprising a populationof stem cells, wherein said stem cells are CD34⁺ and CD44⁻, therebytreating the bone defect in the subject. In certain embodiments, thebone defect is (a) an osteolytic lesion associated with a cancer, (b) abone fracture, or (c) a spine in need of fusion. In certain embodiments,the osteolytic lesion is associated with multiple myeloma, bone cancer,or metastatic cancer. In certain embodiments, the bone fracture is anon-union fracture. In certain embodiments, an implantable compositioncomprising a population of nonadherent stem cells is administered to thesubject. In certain embodiments, the implantable composition issurgically implanted. In certain embodiments, an injectable compositioncomprising a population of nonadherent stem cells is administered to thesubject. In certain embodiments, the injectable composition issurgically administered to the region of the bone defect. In certainembodiments, the injectable composition is systemically administered.

In certain embodiments, the stem cells are CD9⁺, CD54⁺, CD90⁺, orCD166⁺. In certain embodiments, the stem cells are CD9⁺, CD54⁺, CD90⁺,and CD166⁺. In certain embodiments, the stem cells are CD31⁺, CD117⁺,CD133⁺, or CD200⁺. In certain embodiments, the stem cells are CD31⁺,CD117⁺, CD133⁺, and CD200⁺. In certain embodiments, at least about 70%of said cells are CD34⁺ and CD44⁻ stem cells. In certain embodiments, atleast about 90% of said cells are CD34⁺ and CD44⁻ stem cells. In certainembodiments, the population has been expanded.

In yet another aspect, provided herein is a method for treating bonedefects in a subject, comprising administering to a subject in needthereof an implantable or injectable composition comprising a populationof stem cells, wherein said stem cells are CD34⁻ and, thereby treatingthe bone defect in the subject. In certain embodiments, the bone defectis (a) an osteolytic lesion associated with a cancer, (b) a bonefracture, or (c) a spine in need of fusion. In certain embodiments, theosteolytic lesion is associated with multiple myeloma, bone cancer, ormetastatic cancer. In certain embodiments, the bone fracture is anon-union fracture. In certain embodiments, an implantable compositioncomprising a population of adherent stem cells is administered to thesubject. In certain embodiments, the implantable composition issurgically implanted. In certain embodiments, an injectable compositioncomprising a population of adherent stem cells is administered to thesubject. In certain embodiments, the injectable composition issurgically administered to the region of the bone defect. In certainembodiments, the injectable composition is systemically administered.

In more specific embodiments of the nonadherent placental stem cells,the isolated CD200⁺, HLA-G⁺ stem cell is CD34⁻, CD38⁻, CD45⁻, CD73⁺ andCD105⁺; the isolated CD73⁺, CD105⁺, and CD200⁺ stem cell is CD34⁻,CD38⁻, CD45⁻, and HLA-G⁺; the isolated CD200⁺, OCT-4⁺ stem cell isCD34⁻, CD38⁻, CD45⁻, CD73⁺, CD105⁺ and HLA-G⁺; the isolated stem cell ofclaim 1, wherein said CD73⁺, CD105⁺ and HLA-G⁺ stem cell is CD34⁻,CD45⁻, OCT-4⁺ and CD200⁺; the isolated CD73⁺ and CD105⁺ stem cell thatfacilitates the formation of one or more embryoid-like bodies is OCT4⁺,CD34⁻, CD38⁻ and CD45⁻; and/or the isolated OCT-4⁺ and which facilitatesthe formation of one or more embryoid-like bodies is CD73⁺, CD105⁺,CD200⁺, CD34⁻, CD38⁻, and CD45⁻. In certain embodiments, the populationcomprises 1×10⁶ said stem cells. In certain embodiments, the populationcomprises 5×10⁶ said stem cells. In certain embodiments, the populationcomprises 1×10⁷ said stem cells. In certain embodiments, the populationcomprises 5×10⁷ said stem cells. In certain embodiments, the populationcomprises 1×10⁸ said stem cells. In certain embodiments, the populationcomprises 5×10⁸ said stem cells. In certain embodiments, the populationcomprises 1×10⁹ said stem cells. In certain embodiments, the populationcomprises 5×10⁹ said stem cells. In certain embodiments, the populationcomprises 1×10¹⁰ said stem cells. In certain embodiments, the stem cellshave been passaged no more than 5 times. In certain embodiments, thestem cells have been passaged no more than 10 times. In certainembodiments, the stem cells have been passaged no more than 15 times. Incertain embodiments, the stem cells have been passaged no more than 20times. In certain embodiments, the population has been expanded.

Also provided herein are methods for producing populations of stem cellsderived from mammalian placenta. In one embodiment, for example,provided herein is a method of producing a cell population comprisingselecting cells that (a) adhere to a substrate, and (b) express CD200and HLA-G; and isolating said cells from other cells to form a cellpopulation. In another embodiment, provided herein is a method ofproducing a cell population, comprising selecting cells that (a) adhereto a substrate, and (b) express CD73, CD105, and CD200; and isolatingsaid cells from other cells to form a cell population. In anotherembodiment, provided herein is a method of producing a cell population,comprising selecting cells that (a) adhere to a substrate and (b)express CD200 and OCT-4; and isolating said cells from other cells toform a cell population. In yet another embodiment, provided herein is amethod of producing a cell population, comprising selecting cells that(a) adhere to a substrate, (b) express CD73 and CD105, and (c)facilitate the formation of one or more embryoid-like bodies whencultured with a population of placental cells under conditions thatallow for the formation of embryoid-like bodies; and isolating saidcells from other cells to form a cell population. In another embodiment,provided herein is a method of producing a cell population, comprisingselecting cells that (a) adhere to a substrate, and (b) express CD73,CD105 and HLA-G; and isolating said cells from other cells to form acell population. Also provided herein is a method of producing a cellpopulation, comprising selecting cells that (a) adhere to a substrate,(b) express OCT-4, and (c) facilitate the formation of one or moreembryoid-like bodies when cultured with a population of placental cellsunder conditions that allow for the formation of embryoid-like bodies;and isolating said cells from other cells to form a cell population. Ina specific embodiment of any of the foregoing methods, said substratecomprises fibronectin. In another specific embodiment, the methodscomprise selecting cells that express ABC-p. In another specificembodiment, the methods comprise selecting cells exhibiting at least onecharacteristic specific to a mesenchymal stem cell. In a more specificembodiment, said characteristic specific to a mesenchymal stem cell isexpression of CD29, expression of CD44, expression of CD90, orexpression of a combination of the foregoing. In another specificembodiment of the methods, said selecting is accomplished using anantibody. In another specific embodiment, said selecting is accomplishedusing flow cytometry. In another specific embodiment, said selecting isaccomplished using magnetic beads. In another specific embodiment, saidselecting is accomplished by fluorescence-activated cell sorting. Inanother specific embodiment of the above methods, said cell populationis expanded.

Also provided herein is a method of producing a stem cell line,comprising transforming a stem cell with a DNA sequence that encodes agrowth-promoting protein; and exposing said stem cell to conditions thatpromote production of said growth-promoting protein. In a specificembodiment, said growth-promoting protein is v-myc, N-myc, c-myc, p53,SV40 large T antigen, polyoma large T antigen, Ela adenovirus or humanpapillomavirus E7 protein. In a more specific embodiment, said DNAsequence is regulatable. In more specific embodiment, said DNA sequenceis regulatable by tetracycline. In another specific embodiment, saidgrowth-promoting protein has a regulatable activity. In another specificembodiment, said growth-promoting protein is a temperature-sensitivemutant.

Also provided herein are cryopreserved stem cell populations. Forexample, provided herein is a population of CD200⁺, HLA-G⁺ stem cells,wherein said cells have been cryopreserved, and wherein said populationis contained within a container. Also provided herein is a population ofCD73⁺, CD105⁺, CD200⁺ stem cells, wherein said stem cells have beencryopreserved, and wherein said population is contained within acontainer. Also provided herein is a population of CD200⁺, OCT-4⁺ stemcells, wherein said stem cells have been cryopreserved, and wherein saidpopulation is contained within a container. Also provided herein is apopulation of CD73⁺, CD105⁺ stem cells, wherein said cells have beencryopreserved, and wherein said population is contained within acontainer, and wherein said stem cells facilitate the formation of oneor more embryoid-like bodies when cultured with a population ofplacental cells under conditions that allow for the formation ofembryoid-like bodies. Further provided herein is a population of CD73⁺,CD105⁺, HLA-G⁺ stem cells, wherein said cells have been cryopreserved,and wherein said population is contained within a container. Alsoprovided herein is a population of OCT-4⁺ stem cells, wherein said cellshave been cryopreserved, wherein said population is contained within acontainer, and wherein said stem cells facilitate the formation of oneor more embryoid-like bodies when cultured with a population ofplacental cells under conditions that allow for the formation ofembryoid-like bodies. In a specific embodiment of any of the foregoingcryopreserved populations, said container is a bag. In various specificembodiments, said population comprises about, at least, or at most 1×10⁶said stem cells, 5×10⁶ said stem cells, 1×10⁷ said stem cells, 5×10⁷said stem cells, 1×10⁸ said stem cells, 5×10⁸ said stem cells, 1×10⁹said stem cells, 5×10⁹ said stem cells, or 1×10¹⁰ said stem cells. Inother specific embodiments of any of the foregoing cryopreservedpopulations, said stem cells have been passaged about, at least, or nomore than 5 times, no more than 10 times, no more than 15 times, or nomore than 20 times. In another specific embodiment of any of theforegoing cryopreserved populations, said stem cells have been expandedwithin said container.

Further provided herein is a method for preparing a mineralized collagenmatrix, comprising mineralizing collagen and crosslinking themineralized collagen matrix. In certain embodiments, the collagen isplacental collagen. In certain embodiments, the collagen is mineralizedwith calcium phosphate. In certain embodiments, the collagen iscrosslinked with butane diol diglycidyl ether. In certain embodiments,the ratio of calcium phosphate to collagen in the mineralizationreaction is 5:95, 10:90, 15:85, 20:80, 25:75, 30:70, 35:65, 40:60,45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85:15, 90:10, or95:5.

3.1 Definitions

As used herein, the term “SH12” refers to an antibody that binds anepitope on the marker CD105. Thus, cells that are referred to as SH2⁺are CD105⁺.

As used herein, the terms “SH13” and SH4” refer to antibodies that bindepitopes present on the marker CD73. Thus, cells that are referred to asSH3⁺ and/or SH4⁺ are CD73⁺.

As used herein, the term “isolated stem cell” means a stem cell that issubstantially separated from other, non-stem cells of the tissue, e.g.,placenta, from which the stem cell is derived. A stem cell is “isolated”if at least about 50%, 60%, 70%, 80%, 90%, 95%, or at least 99% of thenon-stem cells with which the stem cell is naturally associated areremoved from the stem cell, e.g., during collection and/or culture ofthe stem cell.

As used herein, the term “population of isolated cells” means apopulation of cells that is substantially separated from other cells ofthe tissue, e.g., placenta, from which the population of cells isderived. A stem cell is “isolated” if at least about 50%, 60%, 70%, 80%,90%, 95%, or at least 99% of the cells with which the population ofcells, or cells from which the population of cells is derived, isnaturally associated are removed from the stem cell, e.g., duringcollection and/or culture of the stem cell.

As used herein, the term “placental stem cell” refers to a stem cell orprogenitor cell that is derived from a mammalian placenta, regardless ofmorphology, cell surface markers, or the number of passages after aprimary culture. The term “placental stem cell” as used herein does not,however, refer to a trophoblast. A cell is considered a “stem cell” ifthe cell retains at least one attribute of a stem cell, e.g., a markeror gene expression profile associated with one or more types of stemcells; the ability to replicate at least 10-40 times in culture, theability to differentiate into cells of all three germ layers; the lackof adult (i.e., differentiated) cell characteristics, or the like. Theterms “placental stem cell” and “placenta-derived stem cell” may be usedinterchangeably.

As used herein, “placental perfusate” means perfusion solution that hasbeen passed through at least part of a placenta, e.g., a human placenta,e.g., through the placental vasculature, including a plurality of cellscollected by the perfusion solution during passage through the placenta.

As used herein, “placental perfusate cells” means nucleated cells, e.g.,total nucleated cells, isolated from, or isolatable from, placentalperfusate.

As used herein, a stem cell is “positive” for a particular marker whenthat marker is detectable. For example, a placental stem cell ispositive for, e.g., CD73 because CD73 is detectable on placental stemcells in an amount detectably greater than background (in comparison to,e.g., an isotype control). A cell is also positive for a marker whenthat marker can be used to distinguish the cell from at least one othercell type, or can be used to select or isolate the cell when present orexpressed by the cell.

As used herein, an “osteogenic cell” is a cell that is capable of eitherdepositing hydroxyapatite, the main component of bone, ordifferentiating into a cell that is capable of depositinghydroxyapatite. An “osteogenic cell” is specifically contemplated asencompassing a cell ordinarily referred to as an osteoblast or anosteocyte.

As used herein, a “matrix” refers to a three-dimensional substance thatis characterized by lacunae dispersed throughout the substance. Thelacunae are suitable, for example, for growth of cells, e.g., stemcells, placenta-derived adherent stem cells, and/or osteogenic cells,within the matrix. Exemplary matrices include, but are not limited to, a(3-tricalcium phosphate substrate, a β-tricalcium phosphate-collagensubstrate, a collagen substrate, a calcium phosphate substrate, amineralized human placental collagen substrate, a hyaluronic acidsubstrate, and a ceramic substrate. Preferably, the matrix can bemineralized by an osteogenic cell present in the lacunae of the matrix.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Viability of placental stem cells from perfusion (A), amnion(B), chorion (C), amnion-chorion plate (D) or umbilical cord (E).Numbers on X-axis designate placenta from which stem cells wereobtained.

FIG. 2: Percent HLA ABC⁻/CD45⁻/CD34⁻/CD133⁺ cells from perfusion (A),amnion (B), chorion (C), amnion-chorion plate (D) or umbilical cord (E)as determined by FACSCalibur. Numbers on X-axis designate placenta fromwhich stem cells were obtained.

FIG. 3: Percent HLA ABC⁻/CD45⁻/CD34⁻/CD133⁺ cells from perfusion (A),amnion (B), chorion (C), amnion-chorion plate (D) or umbilical cord (E),as determined by FACS Aria. Numbers on X-axis designate placenta fromwhich stem cells were obtained.

FIG. 4: HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105, CD117, CD200expression in stem cells derived from placental perfusate.

FIG. 5: HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105, CD117, CD200expression in stem cells derived from amnion.

FIG. 6: HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105, CD117, CD200expression in stem cells derived from chorion.

FIG. 7: HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105, CD117, CD200expression in stem cells derived from amnion-chorion plate.

FIG. 8: HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105, CD117, CD200expression in stem cells derived from umbilical cord.

FIG. 9: Average expression of HLA-G, CD10, CD13, CD33, CD38, CD44, CD90,CD105, CD117, CD200 expression in stem cells derived from perfusion (A),amnion (B), chorion (C), amnion-chorion plate (D) or umbilical cord (E).

FIG. 10: Culture time courses for amnion/chorion (AC), umbilical cord(UC), bone marrow-derived stem cell (BM-MSC) and human dermal fibroblast(NHDF) cell lines used in this study. All cultures were grown andpropagated using the same seeding and passage densities. Circlesindicate which cultures were used for RNA isolation. Late cultures wereharvested just prior to senescence. Two UC cultures were harvested at 38doublings (UC-38) to compare the effect of trypsinization on geneexpression. All other cultures were lysed directly in their cultureflasks prior to RNA isolation.

FIG. 11: Line plot of relative expression levels of 8215 genes inamnion/chorion (AC), umbilical cord (UC), bone marrow-derived stem cell(BM-MSC) and human dermal fibroblast (DF) cells. The number associatedwith each cell line designation on the X-axis indicates the number ofdays the cell line was cultured prior to evaluation of gene expressionlevels. The chart was generated from RNA expression data analyzed byGeneSpring software. AC-03 was used as the selected condition.

FIG. 12: Subset of the all genes list showing genes over-expressed6-fold in AC-03 for amnion/chorion (AC), umbilical cord (UC), bonemarrow-derived stem cell (BM-MSC) and human dermal fibroblast (DF)cells. The number associated with each cell line designation on theX-axis indicates the number of days the cell line was cultured prior toevaluation of gene expression levels. The chart was generated from RNAexpression data analyzed by GeneSpring software. AC-03 was used as theselected condition.

FIG. 13: Placental stem cell-specific or umbilical cord stemcell-specific genes found by fold change filtering for amnion/chorion(AC), umbilical cord (UC), bone marrow-derived stem cell (BM-MSC) andhuman dermal fibroblast (DF) cells. The number associated with each cellline designation on the X-axis indicates the number of days the cellline was cultured prior to evaluation of gene expression levels. Thechart was generated from RNA expression data analyzed by GeneSpringsoftware. AC-03 was used as the selected condition.

FIG. 14A-B: Alkaline phosphate activity of both placental stem cells(FIG. 14A) and mesenchymal stem cells (FIG. 14B) cultured in twodifferent media formulations.

FIG. 15A-B: Mineralization of placental stem cells (FIG. 15A) culturedin two different media formulations. FIG. 15B shows the amount ofcalcium recovered from cell layers induced with OS medium compared tothose cultured in AnthrolB medium.

FIG. 16A-B: Deposits of minerals by placental stem cells induced in OSmedium (FIG. 16B), but not in AnthrolB medium (FIG. 16A).

FIG. 17: Time course of growth of placental stem cells and mesenchymalstem cells grown on two different scaffolds.

FIG. 18: Scanning electron micrographs (20×) of placental stem cells andmesenchymal stem cells grown in OS medium and AnthrolB on a β-tricalciumphosphate substrate.

FIG. 19: Scanning electron micrographs (5000×) of placental stem cellsand mesenchymal stem cells grown in OS medium and AnthrolB on aβ-tricalcium phosphate substrate.

FIG. 20A-D: Alizarin red staining of mesenchymal stem cells (FIGS. 20Aand 20B) and stem cells obtained from human perfused placenta cells(FIGS. 20C and 20D) showing calcium mineralization following culture inOS medium, but not DMEM.

FIG. 21: AP activity of mesenchymal stem cells and stem cells following10 days culturing in OS medium in the presence of a β-tricalciumphosphate substrate.

FIG. 22A-B: Electromicrographs showing collagen fibrils (FIG. 22A) andmineralized collagen fibrils (FIG. 22B).

FIG. 23: Diagram showing that the final mineral/collagen ratio ofcrosslinked mineralized collagen was close to the input mineral/collagenratio.

FIG. 24: Histological section of cranial defect 3 weekspost-implantation. Massive deposition of bond within the defect can beseen in the placental stem cell-HEALOS™ explant.

FIGS. 25A-25C: X-ray analysis of cranial defects at 7 weekspost-implantation. FIG. 25A: arrow indicates positive control explantBMP-2+HEALOS™ FIG. 25B: arrow indicates placental stem cell+HEALOS™explant showing bone deposition. FIG. 25C: Negative controls HEALOS™alone and cranial defect without explant.

FIG. 26: Quantification of bone formation by densitometry. Increasinggrayscale (Y axis) indicates increasing bone density/deposition. X axis:Treatment class.

5. DETAILED DESCRIPTION 5.1 Placental Stem Cells and Placental Stem CellPopulations

Placental stem cells are stem cells, obtainable from a placenta or partthereof, that adhere to a tissue culture substrate and have the capacityto differentiate into non-placental cell types. Placental stem cells canbe either fetal or maternal in origin (that is, can have the genotype ofeither the mother or fetus). Populations of placental stem cells, orpopulations of cells comprising placental stem cells, can compriseplacental stem cells that are solely fetal or maternal in origin, or cancomprise a mixed population of placental stem cells of both fetal andmaternal origin. The placental stem cells, and populations of cellscomprising the placental stem cells, can be identified and selected bythe morphological, marker, and culture characteristic discussed below.

5.1.1 Physical and Morphological Characteristics

The nonadherent, CD34⁺ stem cells provided herein, when cultured inprimary cultures or in cell culture, do not typically adhere to thetissue culture substrate. The nonadherent stem cells in culturetypically appear rounded, similar to CD34⁺ stem cells from bone marrowor peripheral blood.

The adherent placental stem cells provided herein, when cultured inprimary cultures or in cell culture, adhere to the tissue culturesubstrate, e.g., tissue culture container surface (e.g., tissue cultureplastic). Placental stem cells in culture assume a generallyfibroblastoid, stellate appearance, with a number of cyotplasmicprocesses extending from the central cell body. The placental stem cellsare, however, morphologically differentiable from fibroblasts culturedunder the same conditions, as the placental stem cells exhibit a greaternumber of such processes than do fibroblasts. Morphologically, placentalstem cells are also differentiable from hematopoietic stem cells, whichgenerally assume a more rounded, or cobblestone, morphology in culture.

5.1.2 Cell Surface, Molecular and Genetic Markers

Nonadherent Placental Stem Cells: In one embodiment, provided herein isan isolated placental stem cell that is nonadherent. In certainembodiments, the isolated stem cell is CD34⁺. In certain embodiments,the isolated stem cell is CD44⁻. In certain embodiments, the isolatedstem cell is CD34⁺ and CD44⁻. In certain embodiments, the isolated stemcell is CD9⁺, CD54⁺, CD90⁺, or CD166⁺. In certain embodiments, theisolated stem cell is CD9⁺, CD54⁺, CD90⁺, and CD166⁺. In certainembodiments, the isolated stem cell is CD31⁺, CD117⁺, CD133⁺, or CD200⁺.In certain embodiments, the isolated stem cell is CD31⁺, CD117⁺, CD133⁺,and CD200⁺. In certain embodiments, the isolated stem cell has beenisolated from a human placenta by perfusion, or by physical orbiochemical disruption of placental tissue, e.g., enzymatic digestion.In certain embodiments, the isolated stem cell has been isolated from ahuman placenta by perfusion. In certain embodiments, the isolated stemcell facilitates formation of a mineralized matrix in a population ofplacental cells when said population is cultured under conditions thatallow the formation of a mineralized matrix.

In another embodiment, provided herein is a population of isolatedplacental cells that are nonadherent. In certain embodiments, thepopulation comprises stem cells that are CD34⁺. In certain embodiments,the population comprises nonadherent stem cells that are CD44⁻. Incertain embodiments, the population comprises stem cells that are CD34⁺and CD44⁻. In certain embodiments, the population comprises stem cellsthat are CD9⁺, CD54⁺, CD90⁺, or CD166⁺. In certain embodiments, thepopulation comprises stem cells that are CD9⁺, CD54⁺, CD90⁺, and CD166⁺.In certain embodiments, the population comprises stem cells that areCD31⁺, CD117⁺, CD133⁺, or CD200⁺. In certain embodiments, the populationcomprises stem cells that are CD31⁺, CD117⁺, CD133⁺, and CD200⁺. Incertain embodiments, the population comprises stem cells, wherein atleast about 70% of said cells are CD34⁺ and CD44⁻ stem cells. In certainembodiments, the population comprises stem cells, wherein at least about90% of said cells are CD34⁺ and CD44⁻ stem cells.

In another aspect, provided herein is a population of isolated placentalstem cells that are CD34⁺ and CD44⁻. In certain embodiments, the stemcells are CD9⁺, CD54⁺, CD90⁺, or CD166⁺. In certain embodiments, thestem cells are CD9⁺, CD54⁺, CD90⁺, and CD166⁺. In certain embodiments,the stem cells are CD31⁺, CD117⁺, CD133⁺, or CD200⁺. In certainembodiments, the stem cells are CD31⁺, CD117⁺, CD133⁺, and CD200⁺. Incertain embodiments, at least about 70% of the stem cells are CD34⁺ andCD44⁻ stem cells. In certain embodiments, at least about 90% of the stemcells are CD34⁺ and CD44⁻ stem cells.

Adherent Placental Stem Cells: Adherent placental stem cells providedherein, and populations of placental stem cells, express a plurality ofmarkers that can be used to identify and/or isolate the stem cells, orpopulations of cells that comprise the stem cells. The adherentplacental stem cells, and stem cell populations provided herein (thatis, two or more placental stem cells) include stem cells and stemcell-containing cell populations obtained directly from the placenta, orany part thereof (e.g., amnion, chorion, placental cotyledons, umbilicalcord, and the like). Placental stem cell populations also includespopulations of (that is, two or more) adherent placental stem cells inculture, and a population in a container, e.g., a bag. Placental stemcells are not, however, trophoblasts.

Adherent placental stem cells provided herein generally express themarkers CD73, CD105, CD200, HLA-G, and/or OCT-4, and do not expressCD34, CD38, or CD45. Placental stem cells can also express HLA-ABC(MHC-1) and HLA-DR. These markers can be used to identify placental stemcells, and to distinguish placental stem cells from other stem celltypes. Because the placental stem cells can express CD73 and CD105, theycan have mesenchymal stem cell-like characteristics. However, becausethe placental stem cells can express CD200 and HLA-G, a fetal-specificmarker, they can be distinguished from mesenchymal stem cells, e.g.,bone marrow-derived mesenchymal stem cells, which express neither CD200nor HLA-G. In the same manner, the lack of expression of CD34, CD38and/or CD45 identifies the placental stem cells as non-hematopoieticstem cells. However, certain subsets of placental stem cells canexpress, for example, CD34, and still be considered a placental stemcell as provided herein.

Thus, in one embodiment, provided herein is an isolated adherentplacental stem cell that is CD200⁺ or HLA-G⁺. In a specific embodiment,said stem cell is a placental stem cell. In a specific embodiment, thestem cell is CD200⁺ and HLA-G⁺. In a specific embodiment, said stem cellis CD73⁺ and CD105⁺. In another specific embodiment, said stem cell isCD34⁻, CD38⁻ or CD45⁻. In another specific embodiment, said stem cell isCD34⁻, CD38⁻ and CD45⁻. In another specific embodiment, said stem cellis CD34⁻, CD38⁻, CD45⁻, CD73⁺ and CD105⁺. In another specificembodiment, said CD200⁺ or HLA-G⁺ stem cell facilitates the formation ofembryoid-like bodies in a population of placental cells comprising thestem cells, under conditions that allow the formation of embryoid-likebodies.

In another embodiment, also provided herein is a method of selecting aplacental stem cell from a plurality of placental cells, comprisingselecting a CD200 or HLA-G placental cell, whereby said cell is aplacental stem cell. In a specific embodiment, said selecting comprisesselecting a placental cell that is both CD200⁺ and HLA-G⁺. In a specificembodiment, said selecting comprises selecting a placental cell that isalso CD73⁺ and CD105⁺. In another specific embodiment, said selectingcomprises selecting a placental cell that is also CD34⁻, CD38⁻ or CD45⁻.In another specific embodiment, said selecting comprises selecting aplacental cell that is also CD34⁻, CD38⁻ and CD45⁻. In another specificembodiment, said selecting comprises selecting a placental cell that isalso CD34⁻, CD38⁻, CD45⁻, CD73⁺ and CD105⁺. In another specificembodiment, said selecting comprises selecting a placental cell thatalso facilitates the formation of embryoid-like bodies in a populationof placental cells comprising the stem cells, under conditions thatallow the formation of embryoid-like bodies.

In another embodiment, provided herein is an isolated population ofcells comprising isolated CD200⁺, HLA-G⁺ placental stem cells. In aspecific embodiment, said population is a population of placental cells.In another specific embodiment, the population is a population ofisolated CD200⁺, HLA-G⁺ placental stem cells. In various embodiments, atleast about 10%, at least about 20%, at least about 30%, at least about40%, at least about 50%, or at least about 60% of said cells are CD200⁺,HLA-G⁺ stem cells. Preferably, at least about 70% of said cells areCD200⁺, HLA-G⁺ stem cells. More preferably, at least about 90%, 95%, or99% of said cells are CD200⁺, HLA-G⁺ stem cells. In a specificembodiment of the isolated populations, said stem cells are also CD73⁺and CD105⁺. In another specific embodiment, said stem cells are alsoCD34⁻, CD38⁻ or CD45⁻. In a more specific embodiment, said stem cellsare also CD34⁻, CD38⁻, CD45⁻, CD73⁺ and CD105⁺. In another embodiment,said isolated population produces one or more embryoid-like bodies whencultured under conditions that allow the formation of embryoid-likebodies.

In another embodiment, provided herein is a method of selecting aplacental stem cell population from a plurality of placental cells,comprising selecting a population of placental cells wherein at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50% at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, or at least about 95% of said cells areCD200⁺, HLA-G⁺ stem cells. In a specific embodiment, said selectingcomprises selecting stem cells that are also CD73⁺ and CD105⁺. Inanother specific embodiment, said selecting comprises selecting stemcells that are also CD34⁻, CD38⁻ or CD45⁻. In another specificembodiment, said selecting comprises selecting stem cells that are alsoCD34⁻, CD38⁻, CD45⁻, CD73⁺ and CD105⁺. In another specific embodiment,said selecting also comprises selecting a population of placental stemcells that forms one or more embryoid-like bodies when cultured underconditions that allow the formation of embryoid-like bodies.

In another embodiment, provided herein is an isolated stem cell that isCD73⁺, CD105⁺, and CD200⁺. In an specific embodiment, said isolated stemcell is an isolated adherent placental stem cell. In another specificembodiment, said stem cell is HLA-G⁺. In another specific embodiment,said stem cell is CD34⁻, CD38⁻ or CD45⁻. In another specific embodiment,said stem cell is CD34⁻, CD38⁻ and CD45⁻. In a more specific embodiment,said stem cell is CD34⁻, CD38⁻, CD45⁻, and HLA-G⁺. In another specificembodiment, the isolated CD73⁺, CD105⁺, and CD200⁺ stem cell facilitatesthe formation of one or more embryoid-like bodies in a population ofplacental cells comprising the stem cell, when the population iscultured under conditions that allow the formation of embryoid-likebodies.

In another embodiment, provided herein is provides a method of selectinga placental stem cell from a plurality of placental cells, comprisingselecting a CD73⁺, CD105⁺, and CD200⁺ placental cell, whereby said cellis a placental stem cell. In a specific embodiment, said selectingcomprises selecting a placental cell that is also HLA-G⁺. In anotherspecific embodiment, said selecting comprises selecting a placental cellthat is also CD34⁻, CD38⁻ or CD45⁻. In another specific embodiment, saidselecting comprises selecting a placental cell that is also CD34⁻, CD38⁻and CD45⁻. In another specific embodiment, said selecting comprisesselecting a placental cell that is also CD34⁻, CD38⁻, CD45⁻, and HLA-G⁺.In another specific embodiment, said selecting additionally comprisesselecting a CD73⁺, CD105⁺, and CD200⁺ stem cell that facilitates theformation of one or more embryoid-like bodies in a population ofplacental cells comprising the stem cell, when the population iscultured under conditions that facilitate formation of embryoid-likebodies.

In another embodiment, provided herein is an isolated population ofcells comprising CD73⁺, CD105⁺, CD200⁺ stem cells. In a specificembodiment, said stem cells are placental stem cells. In anotherspecific embodiment, the population is a population of CD73⁺, CD105⁺,CD200⁺ isolated placental stem cells. In various embodiments, at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, or at least about 60% of said cells are CD73⁺,CD105⁺, CD200⁺ stem cells. In another embodiment, at least about 70% ofsaid cells in said population of cells are CD73⁺, CD105⁺, CD200⁺ stemcells. In another embodiment, at least about 90%, 95% or 99% of saidcells in said population of cells are CD73⁺, CD105⁺, CD200⁺ stem cells.In a specific embodiment of said populations, said stem cells areHLA-G⁺. In another specific embodiment, said stem cells are CD34⁻, CD38⁻or CD45⁻. In another specific embodiment, said stem cells are CD34⁻,CD38⁻ and CD45⁻. In a more specific embodiment, said stem cells areCD34⁻, CD38⁻, CD45⁻, and HLA-G⁺. In another specific embodiment, saidpopulation of cells produces one or more embryoid-like bodies whencultured under conditions that allow the formation of embryoid-likebodies.

In another embodiment, provided herein is a method of selecting aplacental stem cell population from a plurality of placental cells,comprising selecting a population of placental cells wherein at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50% at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, or at least about 95% of said cells areCD73⁺, CD105⁺, CD200⁺ stem cells. In a specific embodiment, saidselecting comprises selecting stem cells that are also HLA-G⁺. Inanother specific embodiment, said selecting comprises selecting stemcells that are also CD34⁻, CD38⁻ or CD45⁻. In another specificembodiment, said selecting comprises selecting stem cells that are alsoCD34⁻, CD38⁻ and CD45⁻. In another specific embodiment, said selectingcomprises selecting stem cells that are also CD34⁻, CD38⁻, CD45⁻, andHLA-G⁺. In another specific embodiment, said selecting additionallycomprises selecting a population of placental cells that produces one ormore embryoid-like bodies when the population is cultured underconditions that allow the formation of embryoid-like bodies.

Also provided herein is an isolated stem cell that is CD200⁺ and OCT-4⁺.In a specific embodiment, the stem cell is CD73⁺ and CD105⁺. In aspecific embodiment, the stem cell is a placental stem cell. In anotherspecific embodiment, said stem cell is HLA-G⁺. In another specificembodiment, said stem cell is CD34⁻, CD38⁻ or CD45⁻. In another specificembodiment, said stem cell is CD34⁻, CD38⁻ and CD45⁻. In a more specificembodiment, said stem cell is CD34⁻, CD38⁻, CD45⁻, CD73⁺, CD105⁺ andHLA-G⁺. In another specific embodiment, the stem cell facilitates theproduction of one or more embryoid-like bodies by a population ofplacental cells that comprises the stem cell, when the population iscultured under conditions that allow the formation of embryoid-likebodies.

In another embodiment, provided herein is a method of selecting aplacental stem cell from a plurality of placental cells, comprisingselecting a CD200⁺ and OCT-4⁺ placental cell, whereby said cell is aplacental stem cell. In a specific embodiment, said selecting comprisesselecting a placental cell that is also HLA-G⁺. In another specificembodiment, said selecting comprises selecting a placental cell that isalso CD34⁻, CD38⁻ or CD45⁻. In another specific embodiment, saidselecting comprises selecting a placental cell that is also CD34⁻, CD38⁻and CD45⁻. In another specific embodiment, said selecting comprisesselecting a placental cell that is also CD34⁻, CD38⁻, CD45⁻, CD73⁺,CD105⁺ and HLA-G⁺. In another specific embodiment, said selectingcomprises selecting a placental stem cell that also facilitates theproduction of one or more embryoid-like bodies by a population ofplacental cells that comprises the stem cell, when the population iscultured under conditions that allow the formation of embryoid-likebodies.

Also provided herein is an isolated population of cells comprisingCD200⁺, OCT-4⁺ stem cells. In a specific embodiment, the stem cells areplacental stem cells. In another specific embodiment, the population isa population of CD200⁺, OCT-4⁺ stem cells. In various embodiments, atleast about 10%, at least about 20%, at least about 30%, at least about40%, at least about 50%, or at least about 60% of said cells are CD200⁺,OCT-4⁺ stem cells. In another embodiment, at least about 70% of saidcells are said CD200⁺, OCT-4⁺ stem cells. In another embodiment, atleast about 90%, 95%, or 99% of said cells are said CD200⁺, OCT-4⁺ stemcells. In a specific embodiment of the isolated populations, said stemcells are CD73⁺ and CD105⁺. In another specific embodiment, said stemcells are HLA-G⁺. In another specific embodiment, said stem cells areCD34⁻, CD38⁻ and CD45⁻. In a more specific embodiment, said stem cellsare CD34⁻, CD38⁻, CD45⁻, CD73⁺, CD105⁺ and HLA-G⁺. In another specificembodiment, the population produces one or more embryoid-like bodieswhen cultured under conditions that allow the formation of embryoid-likebodies.

In another embodiment, provided herein is a method of selecting aplacental stem cell population from a plurality of placental cells,comprising selecting a population of placental cells wherein at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50% at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, or at least about 95% of said cells areCD200⁺, OCT-4⁺ stem cells. In a specific embodiment, said selectingcomprises selecting stem cells that are also CD73⁺ and CD105⁺. Inanother specific embodiment, said selecting comprises selecting stemcells that are also HLA-G⁺. In another specific embodiment, saidselecting comprises selecting stem cells that are also CD34⁻, CD38⁻ andCD45⁻. In another specific embodiment, said stem cells are also CD34⁻,CD38⁻, CD45⁻, CD73⁺, CD105⁺ and HLA-G⁺.

Further provided herein is an isolated stem cell that is CD73⁺, CD105⁺and HLA-G⁺. In a specific embodiment, the stem cell is a placental stemcell. In another specific embodiment, said stem cell is CD34⁻, CD38⁻ orCD45⁻. In another specific embodiment, said stem cell is CD34⁻, CD38⁻and CD45⁻. In another specific embodiment, said stem cell is OCT-4⁺. Inanother specific embodiment, said stem cell is CD200⁺. In a morespecific embodiment, said stem cell is CD34⁻, CD38⁻, CD45⁻, OCT-4⁺ andCD200⁺. In another specific embodiment, said stem cell facilitates theformation of embryoid-like bodies in a population of placental cellscomprising said stem cell, when the population is cultured underconditions that allow the formation of embryoid-like bodies.

In another embodiment, also provided herein is a method of selecting aplacental stem cell from a plurality of placental cells, comprisingselecting a CD73⁺, CD105⁺ and HLA-G⁺ placental cell, whereby said cellis a placental stem cell. In a specific embodiment, said selectingcomprises selecting a placental cell that is also CD34⁻, CD38⁻ or CD45⁻.In another specific embodiment, said selecting comprises selecting aplacental cell that is also CD34⁻, CD38⁻ and CD45⁻. In another specificembodiment, said selecting comprises selecting a placental cell that isalso OCT-4⁺. In another specific embodiment, said selecting comprisesselecting a placental cell that is also CD200⁺. In another specificembodiment, said selecting comprises selecting a placental cell that isalso CD34⁻, CD38⁻, CD45⁻, OCT-4⁺ and CD200⁺. In another specificembodiment, said selecting comprises selecting a placental cell thatalso facilitates the formation of one or more embryoid-like bodies in apopulation of placental cells that comprises said stem cell, when saidpopulation is culture under conditions that allow the formation ofembryoid-like bodies.

Also provided herein is an isolated population of cells comprisingCD73⁺, CD105⁺ and HLA-G⁺ stem cells. In a specific embodiment, said stemcells are placental stem cells. In another specific embodiment, saidpopulation is a population of CD73⁺, CD105⁺ and HLA-G⁺ stem cells. Invarious embodiments, at least about 10%, at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, or at least about 60%of said cells are CD73⁺, CD105⁺ and HLA-G⁺ stem cells. In anotherembodiment, at least about 70% of said cells are CD73⁺, CD105⁺ andHLA-G⁺. In another embodiment, at least about 90%, 95% or 99% of saidcells are CD73⁺, CD105⁺ and HLA-G⁺ stem cells. In a specific embodimentof the above populations, said stem cells are CD34⁻, CD38⁻ or CD45⁻. Inanother specific embodiment, said stem cells are CD34⁻, CD38⁻ and CD45⁻.In another specific embodiment, said stem cells are OCT-4⁺. In anotherspecific embodiment, said stem cells are CD200⁺. In a more specificembodiment, said stem cells are CD34⁻, CD38⁻, CD45⁻, OCT-4⁺ and CD200⁺.In another embodiment, provided herein is a method of selecting aplacental stem cell population from a plurality of placental cells,comprising selecting a population of placental cells wherein a majorityof said cells are CD73⁺, CD105⁺ and HLA-G⁺. In a specific embodiment,said majority of cells are also CD34⁻, CD38⁻ or CD45⁻. In anotherspecific embodiment, said majority of cells are also CD34⁻, CD38⁻ andCD45⁻. In another specific embodiment, said majority of cells are alsoCD200⁺. In another specific embodiment, said majority of cells are alsoCD34⁻, CD38⁻, CD45⁻, OCT-4⁺ and CD200⁺.

In another embodiment, provided herein is an isolated stem cell that isCD73⁺ and CD105⁺ and which facilitates the formation of one or moreembryoid-like bodies in a population of isolated placental cellscomprising said stem cell when said population is cultured underconditions that allow formation of embryoid-like bodies. In a specificembodiment, said stem cell is CD34⁻, CD38⁻ or CD45⁻. In another specificembodiment, said stem cell is CD34⁻, CD38⁻ and CD45⁻. In anotherspecific embodiment, said stem cell is OCT4⁺. In a more specificembodiment, said stem cell is OCT4⁺, CD34⁻, CD38⁻ and CD45⁻.

Further provided herein is a population of isolated placental cellscomprising CD73⁺, CD105⁺ stem cells, wherein said population forms oneor more embryoid-like bodies under conditions that allow formation ofembryoid-like bodies. In a specific embodiment, said stem cell is aplacental stem cell. In another specific embodiment, said population isa population of placental stem cells that are CD73⁺, CD105⁺ stem cells,wherein said population forms one or more embryoid-like bodies underconditions that allow formation of embryoid-like bodies. In variousembodiments, at least about 10%, at least about 20%, at least about 30%,at least about 40%, at least about 50% at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, or at least about 95%of said isolated placental cells are CD73⁺, CD105⁺ stem cells. In aspecific embodiment of the above populations, said stem cells are CD34⁻,CD38⁻ or CD45⁻. In another specific embodiment, said stem cells areCD34⁻, CD38⁻ and CD45⁻. In another specific embodiment, said stem cellsare OCT-4⁺. In a more specific embodiment, said stem cells are OCT-4⁺,CD34⁻, CD38⁻ and CD45⁻. In other specific embodiments, said populationhas been expanded, for example, has been passaged at least once, atleast three times, at least five times, at least 10 times, at least 15times, or at least 20 times.

Further provided herein is an isolated stem cell that is OCT-4⁺ andwhich facilitates formation of one or more embryoid-like bodies in apopulation of isolated placental cells comprising said stem cell whencultured under conditions that allow formation of embryoid-like bodies.In a specific embodiment, said stem cell is CD73⁺ and CD105⁺. In anotherspecific embodiment, said stem cell is CD34⁻, CD38⁻, or CD45⁻. Inanother specific embodiment, said stem cell is CD200⁺. In a morespecific embodiment, said stem cell is CD73⁺, CD105⁺, CD200⁺, CD34⁻,CD38⁻, and CD45⁻.

Also provided herein is a population of isolated placental cellscomprising OCT-4⁺ stem cells, wherein said population forms one or moreembryoid-like bodies when cultured under conditions that allow theformation of embryoid-like bodies. In a specific embodiment, the stemcells are placental stem cells. In another specific embodiment, saidpopulation is a population of placental stem cells that are OCT-4⁺ stemcells, wherein said population forms one or more embryoid-like bodieswhen cultured under conditions that allow the formation of embryoid-likebodies. In various embodiments, at least about 10%, at least about 20%,at least about 30%, at least about 40%, at least about 50% at leastabout 60%, at least about 70%, at least about 80%, at least about 90%,or at least about 95% of said isolated placental cells are OCT4⁺ stemcells. In a specific embodiment of the above populations, said stemcells are CD73⁺ and CD105⁺. In another specific embodiment, said stemcells are CD34⁻, CD38⁻, or CD45⁻. In another specific embodiment, saidstem cells are CD200⁺. In a more specific embodiment, said stem cellsare CD73⁺, CD105⁺, CD200⁺, CD34⁻, CD38⁻, and CD45⁻. In another specificembodiment, said population has been expanded, for example, passaged atleast once, at least three times, at least five times, at least 10times, at least 15 times, or at least 20 times.

Further provided herein are placental stem cells that are obtained byenzymatic digestion (see Section 5.2.3) or perfusion (see Section5.2.4). For example, provided herein is an isolated population ofplacental stem cells that is produced according to a method comprisingperfusing a mammalian placenta that has been drained of cord blood andperfused to remove residual blood; perfusing said placenta with aperfusion solution; and collecting said perfusion solution, wherein saidperfusion solution after perfusion comprises a population of placentalcells that comprises placental stem cells; and isolating a plurality ofsaid placental stem cells from said population of cells. In a specificembodiment, the perfusion solution is passed through both the umbilicalvein and umbilical arteries and collected after it exudes from theplacenta. Populations of placental stem cells produced by this methodtypically comprise a mixture of fetal and maternal cells. In anotherspecific embodiment, the perfusion solution is passed through theumbilical vein and collected from the umbilical arteries, or passedthrough the umbilical arteries and collected from the umbilical vein.Populations of placental stem cells produced by this method typicallyare substantially exclusively fetal in origin; that is, e.g., greaterthan 90%, 95%, 99%, or 99.5% of the placental stem cells in thepopulation are fetal in origin.

In various embodiments, the placental stem cells, contained within apopulation of cells obtained from perfusion of a placenta, are at leastabout 50%, 60%, 70%, 80%, 90%, 95%, 99% or at least 99.5% of saidpopulation of placental cells. In another specific embodiment, theplacental stem cells collected by perfusion comprise fetal and maternalcells. In another specific embodiment, the placental stem cellscollected by perfusion are at least about 50%, 60%, 70%, 80%, 90%, 95%,99% or at least 99.5% fetal cells.

In another specific embodiment, provided herein is a compositioncomprising a population of isolated placental stem cells collected byperfusion, wherein said composition comprises at least a portion of theperfusion solution used to collect the placental stem cells.

Further provided herein is an isolated population of the placental stemcells described herein that is produced according to a method comprisingdigesting placental tissue with a tissue-disrupting enzyme to obtain apopulation of placental cells comprising placental stem cells, andisolating a plurality of placental stem cells from the remainder of saidplacental cells. The whole, or any part of, the placenta can be digestedto obtain placental stem cells. In specific embodiments, for example,said placental tissue is a whole placenta, an amniotic membrane,chorion, a combination of amnion and chorion, or a combination of any ofthe foregoing. In other specific embodiment, the tissue-disruptingenzyme is trypsin or collagenase. In various embodiments, the placentalstem cells, contained within a population of cells obtained fromdigesting a placenta, are at least about 50%, 60%, 70%, 80%, 90%, 95%,99% or at least 99.5% of said population of placental cells.

Gene profiling confirms that isolated adherent placental stem cells, andpopulations of isolated placental stem cells, are distinguishable fromother cells, e.g., mesenchymal stem cells, e.g., bone marrow-derivedstem cells. The adherent placental stem cells described herein, can bedistinguished from mesenchymal stem cells on the basis of the expressionof one or more genes, the expression of which is specific to placentalstem cells or umbilical cord stem cells in comparison to bonemarrow-derived mesenchymal stem cells. In particular, adherent placentalstem cells can be distinguished from mesenchymal stem cells on the basisof the expression of one or more gene, the expression of which issignificantly higher (that is, at least twofold higher) in placentalstem cells than in mesenchymal stem cells, wherein the one or more geneis(are) ACTG2, ADARB1, AMIGO2, ATRS-1, B4GALT6, BCHE, C11orf9, CD200,COL4A1, COL4A2, CPA4, DMD, DSC3, DSG2, ELOVL2, F2RL1, FLJ10781, GATA6,GPR126, GPRC5B, ICAM1, IER3, IGFBP7, IL1A, IL6, IL18, KRT18, KRT8, LIPG,LRAP, MATN2, MEST, NFE2L3, NUAK1, PCDH7, PDLIM3, PJP2, RTN1, SERPINB9,ST3GAL6, ST6GALNAC5, SLC12A8, TCF21, TGFB2, VTN, ZC3H12A, or acombination of any of the foregoing, wherein the expression of thesegenes is higher in placental stem cells or umbilical cord stem cellsthan in bone marrow-derived stem cells, when the stem cells are grownunder equivalent conditions. In a specific embodiment, the placentalstem cell-specific or umbilical cord stem cell-specific gene is CD200.

The level of expression of these genes can be used to confirm theidentity of a population of placental cells, to identify a population ofcells as comprising at least a plurality of placental stem cells, or thelike. The population of placental stem cells, the identity of which isconfirmed, can be clonal, e.g., a population of placental stem cellsexpanded form a single placental stem cell, or a mixed population ofstem cells, e.g., a population of cells comprising solely placental stemcells that are expanded from multiple placental stem cells, or apopulation of cells comprising placental stem cells and at least oneother type of cell.

The level of expression of these genes can be used to select populationsof adherent placental stem cells. For example, a population of cells,e.g., clonally-expanded cells, is selected if the expression of one ormore of these genes is significantly higher in a sample from thepopulation of cells than in an equivalent population of mesenchymal stemcells. Such selecting can be of a population from a plurality ofplacental stem cells populations, from a plurality of cell populations,the identity of which is not known, etc.

Adherent placental stem cells can be selected on the basis of the levelof expression of one or more such genes as compared to the level ofexpression in said one or more genes in a mesenchymal stem cell control.In one embodiment, the level of expression of said one or more genes ina sample comprising an equivalent number of mesenchymal stem cells isused as a control. In another embodiment, the control, for placentalstem cells tested under certain conditions, is a numeric valuerepresenting the level of expression of said one or more genes inmesenchymal stem cells under said conditions.

The isolated populations of adherent or nonadherent placental stem cellsdescribed above, and populations of placental stem cells generally, cancomprise about, at least, or no more than, 1×10⁵, 5×10⁵, 1×10⁶, 5×10⁶,1×10⁷, 5×10⁷, 1×10⁸, 5×10⁸, 1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰, 1×10¹¹ or moreplacental stem cells.

5.1.3 Growth in Culture

The growth of the placental stem cells described herein, as for anymammalian cell, depends in part upon the particular medium selected forgrowth. Under optimum conditions, placental stem cells typically doublein number in 3-5 days. During culture, the placental stem cells providedherein adhere to a substrate in culture, e.g. the surface of a tissueculture container (e.g., tissue culture dish plastic, fibronectin-coatedplastic, and the like) and form a monolayer.

Populations of isolated adherent placental cells that comprise theplacental stem cells provided herein, when cultured under appropriateconditions, form embryoid-like bodies, that is, three-dimensionalclusters of cells grow atop the adherent stem cell layer. Cells withinthe embryoid-like bodies express markers associated with very early stemcells, e.g., OCT-4, Nanog, SSEA3 and SSEA4. Cells within theembryoid-like bodies are typically not adherent to the culturesubstrate, as are the placental stem cells described herein, but remainattached to the adherent cells during culture. Embryoid-like body cellsare dependent upon the adherent placental stem cells for viability, asembryoid-like bodies do not form in the absence of the adherent stemcells. The adherent placental stem cells thus facilitate the growth ofone or more embryoid-like bodies in a population of placental cells thatcomprise the adherent placental stem cells. Without wishing to be boundby theory, the cells of the embryoid-like bodies are thought to grow onthe adherent placental stem cells much as embryonic stem cells grow on afeeder layer of cells. Mesenchymal stem cells, e.g., bone marrow-derivedmesenchymal stem cells, do not develop embryoid-like bodies in culture.

5.2 Methods of Obtaining Placental Stem Cells 5.2.1 Stem Cell CollectionComposition

Further provided herein are methods of collecting and isolatingplacental stem cells. Generally, stem cells are obtained from amammalian placenta using a physiologically-acceptable solution, e.g., astem cell collection composition. A stem cell collection composition isdescribed in detail in related U.S. Provisional Application No.60/754,969, entitled “Improved Medium for Collecting Placental StemCells and Preserving Organs,” filed on Dec. 29, 2005.

The stem cell collection composition can comprise anyphysiologically-acceptable solution suitable for the collection and/orculture of stem cells, for example, a saline solution (e.g.,phosphate-buffered saline, Kreb's solution, modified Kreb's solution,Eagle's solution, 0.9% NaCl. etc.), a culture medium (e.g., DMEM,H.DMEM, etc.), and the like.

The stem cell collection composition can comprise one or more componentsthat tend to preserve placental stem cells, that is, prevent theplacental stem cells from dying, or delay the death of the placentalstem cells, reduce the number of placental stem cells in a population ofcells that die, or the like, from the time of collection to the time ofculturing. Such components can be, e.g., an apoptosis inhibitor (e.g., acaspase inhibitor or JNK inhibitor); a vasodilator (e.g., magnesiumsulfate, an antihypertensive drug, atrial natriuretic peptide (ANP),adrenocorticotropin, corticotropin-releasing hormone, sodiumnitroprusside, hydralazine, adenosine triphosphate, adenosine,indomethacin or magnesium sulfate, a phosphodiesterase inhibitor, etc.);a necrosis inhibitor (e.g., 2-(1H-Indol-3-yl)-3-pentylamino-maleimide,pyrrolidine dithiocarbamate, or clonazepam); a TNF-α inhibitor; and/oran oxygen-carrying perfluorocarbon (e.g., perfluorooctyl bromide,perfluorodecyl bromide, etc.).

The stem cell collection composition can comprise one or moretissue-degrading enzymes, e.g., a metalloprotease, a serine protease, aneutral protease, an RNase, or a DNase, or the like. Such enzymesinclude, but are not limited to, collagenases (e.g., collagenase I, II,III or IV, a collagenase from Clostridium histolyticum, etc.); dispase,thermolysin, elastase, trypsin, LIBERASE, hyaluronidase, and the like.

The stem cell collection composition can comprise a bacteriocidally orbacteriostatically effective amount of an antibiotic. In certainnon-limiting embodiments, the antibiotic is a macrolide (e.g.,tobramycin), a cephalosporin (e.g., cephalexin, cephradine, cefuroxime,cefprozil, cefaclor, cefixime or cefadroxil), a clarithromycin, anerythromycin, a penicillin (e.g., penicillin V) or a quinolone (e.g.,ofloxacin, ciprofloxacin or norfloxacin), a tetracycline, astreptomycin, etc. In a particular embodiment, the antibiotic is activeagainst Gram(+) and/or Gram(−) bacteria, e.g., Pseudomonas aeruginosa,Staphylococcus aureus, and the like.

The stem cell collection composition can also comprise one or more ofthe following compounds: adenosine (about 1 mM to about 50 mM);D-glucose (about 20 mM to about 100 mM); magnesium ions (about 1 mM toabout 50 mM); a macromolecule of molecular weight greater than 20,000daltons, in one embodiment, present in an amount sufficient to maintainendothelial integrity and cellular viability (e.g., a synthetic ornaturally occurring colloid, a polysaccharide such as dextran or apolyethylene glycol present at about 25 g/l to about 100 g/1, or about40 g/l to about 60 g/1); an antioxidant (e.g., butylated hydroxyanisole,butylated hydroxytoluene, glutathione, vitamin C or vitamin E present atabout 25 μM to about 100 μM); a reducing agent (e.g., N-acetylcysteinepresent at about 0.1 mM to about 5 mM); an agent that prevents calciumentry into cells (e.g., verapamil present at about 2 μM to about 25 μM);nitroglycerin (e.g., about 0.05 g/L to about 0.2 g/L); an anticoagulant,in one embodiment, present in an amount sufficient to help preventclotting of residual blood (e.g., heparin or hirudin present at aconcentration of about 1000 units/1 to about 100,000 units/1); or anamiloride containing compound (e.g., amiloride, ethyl isopropylamiloride, hexamethylene amiloride, dimethyl amiloride or isobutylamiloride present at about 1.0 μM to about 5 μM).

5.2.2 Collection and Handling of Placenta

Generally, a human placenta is recovered shortly after its expulsionafter birth. In a preferred embodiment, the placenta is recovered from apatient after informed consent and after a complete medical history ofthe patient is taken and is associated with the placenta. Preferably,the medical history continues after delivery. Such a medical history canbe used to coordinate subsequent use of the placenta or the stem cellsharvested therefrom. For example, human placental stem cells can beused, in light of the medical history, for personalized medicine for theinfant associated with the placenta, or for parents, siblings or otherrelatives of the infant.

Prior to recovery of placental stem cells, the umbilical cord blood andplacental blood are removed. In certain embodiments, after delivery, thecord blood in the placenta is recovered. The placenta can be subjectedto a conventional cord blood recovery process. Typically a needle orcannula is used, with the aid of gravity, to exsanguinate the placenta(see, e.g., Anderson, U.S. Pat. No. 5,372,581; Hessel et al., U.S. Pat.No. 5,415,665). The needle or cannula is usually placed in the umbilicalvein and the placenta can be gently massaged to aid in draining cordblood from the placenta. Such cord blood recovery may be performedcommercially, e.g., LifeBank USA, Cedar Knolls, N.J., ViaCord, CordBlood Registry and Cryocell. Preferably, the placenta is gravity drainedwithout further manipulation so as to minimize tissue disruption duringcord blood recovery.

Typically, a placenta is transported from the delivery or birthing roomto another location, e.g., a laboratory, for recovery of cord blood andcollection of stem cells by, e.g., perfusion or tissue dissociation. Theplacenta is preferably transported in a sterile, thermally insulatedtransport device (maintaining the temperature of the placenta between20-28° C.), for example, by placing the placenta, with clamped proximalumbilical cord, in a sterile zip-lock plastic bag, which is then placedin an insulated container. In another embodiment, the placenta istransported in a cord blood collection kit substantially as described inpending U.S. patent application Ser. No. 11/230,760, filed Sep. 19,2005. Preferably, the placenta is delivered to the laboratory four totwenty-four hours following delivery. In certain embodiments, theproximal umbilical cord is clamped, preferably within 4-5 cm(centimeter) of the insertion into the placental disc prior to cordblood recovery. In other embodiments, the proximal umbilical cord isclamped after cord blood recovery but prior to further processing of theplacenta.

The placenta, prior to stem cell collection, can be stored under sterileconditions and at either room temperature or at a temperature of 5 to25° C. (centigrade). The placenta may be stored for a period of longerthan forty eight hours, and preferably for a period of four totwenty-four hours prior to perfusing the placenta to remove any residualcord blood. The placenta is preferably stored in an anticoagulantsolution at a temperature of 5 to 25° C. (centigrade). Suitableanticoagulant solutions are well known in the art. For example, asolution of heparin or warfarin sodium can be used. In a preferredembodiment, the anticoagulant solution comprises a solution of heparin(e.g., 1% w/w in 1:1000 solution). The exsanguinated placenta ispreferably stored for no more than 36 hours before placental stem cellsare collected.

The mammalian placenta or a part thereof, once collected and preparedgenerally as above, can be treated in any art-known manner, e.g., can beperfused or disrupted, e.g., digested with one or more tissue-disruptingenzymes, to obtain stem cells.

5.2.3 Physical Disruption and Enzymatic Digestion of Placental Tissue

In one embodiment, stem cells are collected from a mammalian placenta byphysical disruption, e.g., enzymatic digestion, of the organ. Forexample, the placenta, or a portion thereof, may be, e.g., crushed,sheared, minced, diced, chopped, macerated or the like, while in contactwith the stem cell collection composition provided herein, and thetissue subsequently digested with one or more enzymes. The placenta, ora portion thereof, may also be physically disrupted and digested withone or more enzymes, and the resulting material then immersed in, ormixed into, the stem cell collection composition. Any method of physicaldisruption can be used, provided that the method of disruption leaves aplurality, more preferably a majority, and more preferably at leastabout 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the cells in said organviable, as determined by, e.g., trypan blue exclusion.

The placenta can be dissected into components prior to physicaldisruption and/or enzymatic digestion and stem cell recovery. Forexample, placental stem cells can be obtained from the amnioticmembrane, chorion, umbilical cord, placental cotyledons, or anycombination thereof. Preferably, placental stem cells are obtained fromplacental tissue comprising amnion and chorion. Typically, placentalstem cells can be obtained by disruption of a small block of placentaltissue, e.g., a block of placental tissue that is about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700, 800, 900 or about 1000 cubic millimeters in volume.

A preferred stem cell collection composition comprises one or moretissue-disruptive enzyme(s). Enzymatic digestion preferably uses acombination of enzymes, e.g., a combination of a matrix metalloproteaseand a neutral protease, for example, a combination of collagenase anddispase. In one embodiment, enzymatic digestion of placental tissue usesa combination of a matrix metalloprotease, a neutral protease, and amucolytic enzyme for digestion of hyaluronic acid, such as a combinationof collagenase, dispase, and hyaluronidase or a combination of LIBERASE(Boehringer Mannheim Corp., Indianapolis, Ind.) and hyaluronidase. Otherenzymes that can be used to disrupt placenta tissue include papain,deoxyribonucleases, serine proteases, such as trypsin, chymotrypsin, orelastase. Serine proteases may be inhibited by alpha 2 microglobulin inserum and therefore the medium used for digestion is usually serum-free.EDTA and DNase are commonly used in enzyme digestion procedures toincrease the efficiency of cell recovery. The digestate is preferablydiluted so as to avoid trapping stem cells within the viscous digest.

Any combination of tissue digestion enzymes can be used. Typicalconcentrations for tissue digestion enzymes include, e.g., 50-200 U/mLfor collagenase I and collagenase IV, 1-10 U/mL for dispase, and 10-100U/mL for elastase. Proteases can be used in combination, that is, two ormore proteases in the same digestion reaction, or can be usedsequentially in order to liberate placental stem cells. For example, inone embodiment, a placenta, or part thereof, is digested first with anappropriate amount of collagenase I at 2 mg/ml for 30 minutes, followedby digestion with trypsin, 0.25%, for 10 minutes, at 37° C. Serineproteases are preferably used consecutively following use of otherenzymes.

In another embodiment, the tissue can further be disrupted by theaddition of a chelator, e.g., ethylene glycol bis(2-aminoethylether)-N,N,N′N′-tetraacetic acid (EGTA) or ethylenediaminetetraaceticacid (EDTA) to the stem cell collection composition comprising the stemcells, or to a solution in which the tissue is disrupted and/or digestedprior to isolation of the stem cells with the stem cell collectioncomposition.

It will be appreciated that where an entire placenta, or portion of aplacenta comprising both fetal and maternal cells (for example, wherethe portion of the placenta comprises the chorion or cotyledons), theplacental stem cells collected will comprise a mix of placental stemcells derived from both fetal and maternal sources. Where a portion ofthe placenta that comprises no, or a negligible number of, maternalcells (for example, amnion), the placental stem cells collected willcomprise almost exclusively fetal placental stem cells.

5.2.4 Placental Perfusion

Placental stem cells can also be obtained by perfusion of the mammalianplacenta. Methods of perfusing mammalian placenta to obtain stem cellsare disclosed, e.g., in Hariri, U.S. Application Publication No.2002/0123141, and in related U.S. Provisional Application No.60/754,969, entitled “Improved Medium for Collecting Placental StemCells and Preserving Organs,” filed on Dec. 29, 2005.

Placental stem cells can be collected by perfusion, e.g., through theplacental vasculature, using, e.g., a stem cell collection compositionas a perfusion solution. In one embodiment, a mammalian placenta isperfused by passage of perfusion solution through either or both of theumbilical artery and umbilical vein. The flow of perfusion solutionthrough the placenta may be accomplished using, e.g., gravity flow intothe placenta. Preferably, the perfusion solution is forced through theplacenta using a pump, e.g., a peristaltic pump. The umbilical vein canbe, e.g., cannulated with a cannula, e.g., a TEFLON® or plastic cannula,that is connected to a sterile connection apparatus, such as steriletubing. The sterile connection apparatus is connected to a perfusionmanifold.

In preparation for perfusion, the placenta is preferably oriented (e.g.,suspended) in such a manner that the umbilical artery and umbilical veinare located at the highest point of the placenta. The placenta can beperfused by passage of a perfusion fluid through the placentalvasculature and surrounding tissue. The placenta can also be perfused bypassage of a perfusion fluid into the umbilical vein and collection fromthe umbilical arteries, or passage of a perfusion fluid into theumbilical arteries and collection from the umbilical vein.

In one embodiment, for example, the umbilical artery and the umbilicalvein are connected simultaneously, e.g., to a pipette that is connectedvia a flexible connector to a reservoir of the perfusion solution. Theperfusion solution is passed into the umbilical vein and artery. Theperfusion solution exudes from and/or passes through the walls of theblood vessels into the surrounding tissues of the placenta, and iscollected in a suitable open vessel from the surface of the placentathat was attached to the uterus of the mother during gestation. Theperfusion solution may also be introduced through the umbilical cordopening and allowed to flow or percolate out of openings in the wall ofthe placenta which interfaced with the maternal uterine wall. Placentalcells that are collected by this method, which can be referred to as a“pan” method, are typically a mixture of fetal and maternal cells.

In another embodiment, the perfusion solution is passed through theumbilical veins and collected from the umbilical artery, or is passedthrough the umbilical artery and collected from the umbilical veins.Placental cells collected by this method, which can be referred to as a“closed circuit” method, are typically almost exclusively fetal.

It will be appreciated that perfusion using the pan method, that is,whereby perfusate is collected after it has exuded from the maternalside of the placenta, results in a mix of fetal and maternal cells. As aresult, the cells collected by this method comprise a mixed populationof placental stem cells of both fetal and maternal origin. In contrast,perfusion solely through the placental vasculature in the closed circuitmethod, whereby perfusion fluid is passed through one or two placentalvessels and is collected solely through the remaining vessel(s), resultsin the collection of a population of placental stem cells almostexclusively of fetal origin.

The closed circuit perfusion method can, in one embodiment, be performedas follows. A post-partum placenta is obtained within about 48 hoursafter birth. The umbilical cord is clamped and cut above the clamp. Theumbilical cord can be discarded, or can processed to recover, e.g.,umbilical cord stem cells, and/or to process the umbilical cord membranefor the production of a biomaterial. The amniotic membrane can beretained during perfusion, or can be separated from the chorion, e.g.,using blunt dissection with the fingers. If the amniotic membrane isseparated from the chorion prior to perfusion, it can be, e.g.,discarded, or processed, e.g., to obtain stem cells by enzymaticdigestion, or to produce, e.g., an amniotic membrane biomaterial, e.g.,the biomaterial described in U.S. Application Publication No.2004/0048796. After cleaning the placenta of all visible blood clots andresidual blood, e.g., using sterile gauze, the umbilical cord vesselsare exposed, e.g., by partially cutting the umbilical cord membrane toexpose a cross-section of the cord. The vessels are identified, andopened, e.g., by advancing a closed alligator clamp through the cut endof each vessel. The apparatus, e.g., plastic tubing connected to aperfusion device or peristaltic pump, is then inserted into each of theplacental arteries. The pump can be any pump suitable for the purpose,e.g., a peristaltic pump. Plastic tubing, connected to a sterilecollection reservoir, e.g., a blood bag such as a 250 mL collection bag,is then inserted into the placental vein. Alternatively, the tubingconnected to the pump is inserted into the placental vein, and tubes toa collection reservoir(s) are inserted into one or both of the placentalarteries. The placenta is then perfused with a volume of perfusionsolution, e.g., about 750 ml of perfusion solution. Cells in theperfusate are then collected, e.g., by centrifugation.

In one embodiment, the proximal umbilical cord is clamped duringperfusion, and more preferably, is clamped within 4-5 cm (centimeter) ofthe cord's insertion into the placental disc.

The first collection of perfusion fluid from a mammalian placenta duringthe exsanguination process is generally colored with residual red bloodcells of the cord blood and/or placental blood. The perfusion fluidbecomes more colorless as perfusion proceeds and the residual cord bloodcells are washed out of the placenta. Generally from 30 to 100 ml(milliliter) of perfusion fluid is adequate to initially exsanguinatethe placenta, but more or less perfusion fluid may be used depending onthe observed results.

The volume of perfusion liquid used to collect placental stem cells mayvary depending upon the number of stem cells to be collected, the sizeof the placenta, the number of collections to be made from a singleplacenta, etc. In various embodiments, the volume of perfusion liquidmay be from 50 mL to 5000 mL, 50 mL to 4000 mL, 50 mL to 3000 mL, 100 mLto 2000 mL, 250 mL to 2000 mL, 500 mL to 2000 mL, or 750 mL to 2000 mL.Typically, the placenta is perfused with 700-800 mL of perfusion liquidfollowing exsanguination.

The placenta can be perfused a plurality of times over the course ofseveral hours or several days. Where the placenta is to be perfused aplurality of times, it may be maintained or cultured under asepticconditions in a container or other suitable vessel, and perfused withthe stem cell collection composition, or a standard perfusion solution(e.g., a normal saline solution such as phosphate buffered saline(“PBS”)) with or without an anticoagulant (e.g., heparin, warfarinsodium, coumarin, bishydroxycoumarin), and/or with or without anantimicrobial agent (e.g., β-mercaptoethanol (0.1 mM); antibiotics suchas streptomycin (e.g., at 40-100 μg/ml), penicillin (e.g., at 40 U/ml),amphotericin B (e.g., at 0.5 μg/ml). In one embodiment, an isolatedplacenta is maintained or cultured for a period of time withoutcollecting the perfusate, such that the placenta is maintained orcultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, or 24 hours, or 2 or 3 or more days beforeperfusion and collection of perfusate. The perfused placenta can bemaintained for one or more additional time(s), e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 ormore hours, and perfused a second time with, e.g., 700-800 mL perfusionfluid. The placenta can be perfused 1, 2, 3, 4, 5 or more times, forexample, once every 1, 2, 3, 4, 5 or 6 hours. In a preferred embodiment,perfusion of the placenta and collection of perfusion solution, e.g.,stem cell collection composition, is repeated until the number ofrecovered nucleated cells falls below 100 cells/ml. The perfusates atdifferent time points can be further processed individually to recovertime-dependent populations of cells, e.g., stem cells. Perfusates fromdifferent time points can also be pooled.

Without wishing to be bound by any theory, after exsanguination and asufficient time of perfusion of the placenta, placental stem cells arebelieved to migrate into the exsanguinated and perfused microcirculationof the placenta where they are collected, preferably by washing into acollecting vessel by perfusion. Perfusing the isolated placenta not onlyserves to remove residual cord blood but also provide the placenta withthe appropriate nutrients, including oxygen. The placenta may becultivated and perfused with a similar solution which was used to removethe residual cord blood cells, preferably, without the addition ofanticoagulant agents.

Perfusion according to the methods provided herein results in thecollection of significantly more placental stem cells than the numberobtainable from a mammalian placenta not perfused with said solution,and not otherwise treated to obtain stem cells (e.g., by tissuedisruption, e.g., enzymatic digestion). In this context, “significantlymore” means at least about 10% more. Perfusion yields significantly moreplacental stem cells than, e.g., the number of placental stem cellsobtainable from culture medium in which a placenta, or portion thereof,has been cultured.

Stem cells can be isolated from placenta by perfusion with a solutioncomprising one or more proteases or other tissue-disruptive enzymes. Ina specific embodiment, a placenta or portion thereof (e.g., amnioticmembrane, amnion and chorion, placental lobule or cotyledon, umbilicalcord, or combination of any of the foregoing) is brought to 25-37° C.,and is incubated with one or more tissue-disruptive enzymes in 200 mL ofa culture medium for 30 minutes. Cells from the perfusate are collected,brought to 4° C., and washed with a cold inhibitor mix comprising 5 mMEDTA, 2 mM dithiothreitol and 2 mM beta-mercaptoethanol. The stem cellsare washed after several minutes with a cold (e.g., 4° C.) stem cellcollection composition provided herein.

5.2.5 Placental Perfusate and Placental Perfusate Cells

Placental perfusate, and placental perfusate cells, e.g., totalnucleated cells isolated from placental perfusate, comprise aheterogeneous collection of cells. Typically, placental perfusate, andplacental perfusate cells, are depleted of erythrocytes prior to use.Such depletion can be carried out by known methods of separating redblood cells from nucleated blood cells. In certain embodiment, theplacental perfusate or perfusate cells are cryopreserved. In certainother embodiments, the placental perfusate comprises, or the perfusatecells comprise, only fetal cells, or a combination of fetal cells andmaternal cells.

Typically, placental perfusate from a single placental perfusioncomprises about 100 million to about 500 million nucleated cells. Incertain embodiments, the placental perfusate or perfusate cells compriseCD34⁺ cells, e.g., hematopoietic stem or progenitor cells. Such cellscan, in a more specific embodiment, comprise CD34⁺CD45⁻ stem orprogenitor cells, CD34⁺CD45⁺ stem or progenitor cells, myeloidprogenitors, lymphoid progenitors, and/or erythroid progenitors. Inother embodiments, placental perfusate and placental perfusate cellscomprise adherent placental stem cells, e.g., CD34⁻ stem cells, e.g.,adherent placental stem cells as described in Section 5.1, above. Inother embodiment, the placental perfusate and placental perfusate cellscomprise, e.g., endothelial progenitor cells, osteoprogenitor cells, andnatural killer cells. In certain embodiments, placental perfusate ascollected from the placenta and depleted of erythrocytes, or perfusatecells isolated from such perfusate, comprise about 6-7% natural killercells (CD3⁻, CD56⁺); about 21-22% T cells (CD3⁺); about 6-7% B cells(CD19⁺); about 1-2% endothelial progenitor cells (CD34⁺, CD31⁺); about2-3% neural progenitor cells (nestin⁺); about 2-5% hematopoieticprogenitor cells (CD34⁺); and about 0.5-1.5% adherent placental stemcells (e.g., CD34⁻, CD117⁻, CD105⁺ and CD44⁺), as determined, e.g. byflow cytometry, e.g., by FACS analysis.

The CD34⁺ stem or progenitor cells in human placental perfusate expressdetectably higher levels of angiogenesis-related markers, e.g., CD31,VEGF-R and/or CXCR4 than do an equivalent number of CD34⁺ cells isolatedfrom umbilical cord blood. In certain embodiments, human placentalperfusate mononuclear cells from a single perfusion that are cultured inENDOCULT® medium with VEGF (for growth of CFU-Hill colonies; StemCellTechnologies, Inc.) generate up to about 20, e.g., about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 CFU-Hillcolonies (endothelial cell progenitors). Development of CFU-Hillcolonies in liquid culture can be demonstrated and assessed, e.g., bymeasuring uptake of diacetylated low density lipoprotein (Dil-acLDL) byendothelial progenitor cells obtained from human placental perfusate at,e.g., seven days of culture in ENDOCULT® medium.

Moreover, CD34⁺CD45⁻ cells from human placental perfusate have adetectably higher expression of angiogenesis related markers CD31 and/orVEGFR than CD34⁺CD45⁺ cells.

Typically, placental perfusate and perfusate cells have low expressionof MHC class I compared to umbilical cord blood cells, and are largelynegative for MHC class II markers.

5.2.6 Isolation, Sorting, and Characterization of Placental Stem Cells

Stem cells from mammalian placenta, whether obtained by perfusion orenyzmatic digestion, can initially be purified from (i.e., be isolatedfrom) other cells by Ficoll gradient centrifugation. Such centrifugationcan follow any standard protocol for centrifugation speed, etc. In oneembodiment, for example, cells collected from the placenta are recoveredfrom perfusate by centrifugation at 5000×g for 15 minutes at roomtemperature, which separates cells from, e.g., contaminating debris andplatelets. In another embodiment, placental perfusate is concentrated toabout 200 ml, gently layered over Ficoll, and centrifuged at about1100×g for 20 minutes at 22° C., and the low-density interface layer ofcells is collected for further processing.

Cell pellets can be resuspended in fresh stem cell collectioncomposition, or a medium suitable for stem cell maintenance, e.g., IMDMserum-free medium containing 2 U/ml heparin and 2 mM EDTA (GibcoBRL,N.Y.). The total mononuclear cell fraction can be isolated, e.g., usingLymphoprep (Nycomed Pharma, Oslo, Norway) according to themanufacturer's recommended procedure.

As used herein, “isolating” placental stem cells means to remove atleast about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of thecells with which the stem cells are normally associated in the intactmammalian placenta. A stem cell from an organ is “isolated” when it ispresent in a population of cells that comprises fewer than 50% of thecells with which the stem cell is normally associated in the intactorgan.

Placental cells obtained by perfusion or digestion can, for example, befurther, or initially, isolated by differential trypsinization using,e.g., a solution of 0.05% trypsin with 0.2% EDTA (Sigma, St. Louis Mo.).Differential trypsinization is possible because placental stem cellstypically detach from plastic surfaces within about five minutes whereasother adherent populations typically require more than 20-30 minutesincubation. The detached placental stem cells can be harvested followingtrypsinization and trypsin neutralization, using, e.g., TrypsinNeutralizing Solution (TNS, Cambrex). In one embodiment of isolation ofadherent cells, aliquots of, for example, about 5-10×10⁶ cells areplaced in each of several T-75 flasks, preferably fibronectin-coated T75flasks. In such an embodiment, the cells can be cultured withcommercially available Mesenchymal Stem Cell Growth Medium (MSCGM)(Cambrex), and placed in a tissue culture incubator (37° C., 5% CO₂).After 10 to 15 days, non-adherent cells are removed from the flasks bywashing with PBS. The PBS is then replaced by MSCGM. Flasks arepreferably examined daily for the presence of various adherent celltypes and in particular, for identification and expansion of clusters offibroblastoid cells.

The number and type of cells collected from a mammalian placenta can bemonitored, for example, by measuring changes in morphology and cellsurface markers using standard cell detection techniques such as flowcytometry, cell sorting, immunocytochemistry (e.g., staining with tissuespecific or cell-marker specific antibodies) fluorescence activated cellsorting (FACS), magnetic activated cell sorting (MACS), by examinationof the morphology of cells using light or confocal microscopy, and/or bymeasuring changes in gene expression using techniques well known in theart, such as PCR and gene expression profiling. These techniques can beused, too, to identify cells that are positive for one or moreparticular markers. For example, using antibodies to CD34, one candetermine, using the techniques above, whether a cell comprises adetectable amount of CD34; if so, the cell is CD34⁺. Likewise, if a cellproduces enough OCT-4 RNA to be detectable by RT-PCR, or significantlymore OCT-4 RNA than an adult cell, the cell is OCT-4⁺ Antibodies to cellsurface markers (e.g., CD markers such as CD34) and the sequence of stemcell-specific genes, such as OCT-4, are well-known in the art.

Placental cells, particularly cells that have been isolated by Ficollseparation, differential adherence, or a combination of both, may besorted using a fluorescence activated cell sorter (FACS). Fluorescenceactivated cell sorting (FACS) is a well-known method for separatingparticles, including cells, based on the fluorescent properties of theparticles (Kamarch, 1987, Methods Enzymol, 151:150-165). Laserexcitation of fluorescent moieties in the individual particles resultsin a small electrical charge allowing electromagnetic separation ofpositive and negative particles from a mixture. In one embodiment, cellsurface marker-specific antibodies or ligands are labeled with distinctfluorescent labels. Cells are processed through the cell sorter,allowing separation of cells based on their ability to bind to theantibodies used. FACS sorted particles may be directly deposited intoindividual wells of 96-well or 384-well plates to facilitate separationand cloning.

In one sorting scheme, stem cells from placenta are sorted on the basisof expression of the markers CD34, CD38, CD44, CD45, CD73, CD105, OCT-4and/or HLA-G. This can be accomplished in connection with procedures toselect stem cells on the basis of their adherence properties in culture.For example, an adherence selection stem can be accomplished before orafter sorting on the basis of marker expression. In one embodiment, forexample, cells are sorted first on the basis of their expression ofCD34; CD34⁻ cells are retained, and cells that are CD200⁺HLA-G⁺, areseparated from all other CD34⁻ cells. In another embodiment, cells fromplacenta are based on their expression of markers CD200 and/or HLA-G;for example, cells displaying either of these markers are isolated forfurther use. Cells that express, e.g., CD200 and/or HLA-G can, in aspecific embodiment, be further sorted based on their expression of CD73and/or CD105, or epitopes recognized by antibodies SH2, SH3 or SH4, orlack of expression of CD34, CD38 or CD45. For example, in oneembodiment, placental cells are sorted by expression, or lack thereof,of CD200, HLA-G, CD73, CD105, CD34, CD38 and CD45, and placental cellsthat are CD200⁺, HLA-G⁺, CD73⁺, CD105⁺, CD34⁻, CD38⁻ and CD45⁻ areisolated from other placental cells for further use.

In another embodiment, magnetic beads can be used to separate cells. Thecells may be sorted using a magnetic activated cell sorting (MACS)technique, a method for separating particles based on their ability tobind magnetic beads (0.5-100 μm diameter). A variety of usefulmodifications can be performed on the magnetic microspheres, includingcovalent addition of antibody that specifically recognizes a particularcell surface molecule or hapten. The beads are then mixed with the cellsto allow binding. Cells are then passed through a magnetic field toseparate out cells having the specific cell surface marker. In oneembodiment, these cells can then isolated and re-mixed with magneticbeads coupled to an antibody against additional cell surface markers.The cells are again passed through a magnetic field, isolating cellsthat bound both the antibodies. Such cells can then be diluted intoseparate dishes, such as microtiter dishes for clonal isolation.

Placental stem cells can also be characterized and/or sorted based oncell morphology and growth characteristics. For example, placental stemcells can be characterized as having, and/or selected on the basis of,e.g., a fibroblastoid appearance in culture. Placental stem cells canalso be characterized as having, and/or be selected, on the basis oftheir ability to form embryoid-like bodies. In one embodiment, forexample, placental cells that are fibroblastoid in shape, express CD73and CD105, and produce one or more embryoid-like bodies in culture areisolated from other placental cells. In another embodiment, OCT-4⁺placental cells that produce one or more embryoid-like bodies in cultureare isolated from other placental cells.

In another embodiment, placental stem cells can be identified andcharacterized by a colony forming unit assay. Colony forming unit assaysare commonly known in the art, such as MESEN CULT™ medium (Stem CellTechnologies, Inc., Vancouver British Columbia)

Placental stem cells can be assessed for viability, proliferationpotential, and longevity using standard techniques known in the art,such as trypan blue exclusion assay, fluorescein diacetate uptake assay,propidium iodide uptake assay (to assess viability); and thymidineuptake assay, MTT cell proliferation assay (to assess proliferation).Longevity may be determined by methods well known in the art, such as bydetermining the maximum number of population doubling in an extendedculture.

Placental stem cells can also be separated from other placental cellsusing other techniques known in the art, e.g., selective growth ofdesired cells (positive selection), selective destruction of unwantedcells (negative selection); separation based upon differential cellagglutinability in the mixed population as, for example, with soybeanagglutinin; freeze-thaw procedures; filtration; conventional and zonalcentrifugation; centrifugal elutriation (counter-streamingcentrifugation); unit gravity separation; countercurrent distribution;electrophoresis; and the like.

5.3 Culture of Placental Stem Cells 5.3.1 Culture Media

Isolated placental stem cells, or placental stem cell population, orcells or placental tissue from which placental stem cells grow out, canbe used to initiate, or seed, cell cultures. Cells are generallytransferred to sterile tissue culture vessels either uncoated or coatedwith extracellular matrix or ligands such as laminin, collagen (e.g.,native or denatured), gelatin, fibronectin, ornithine, vitronectin, andextracellular membrane protein (e.g., MATRIGEL (BD Discovery Labware,Bedford, Mass.)).

Placental stem cells can be cultured in any medium, and under anyconditions, recognized in the art as acceptable for the culture of stemcells. Preferably, the culture medium comprises serum. Placental stemcells can be cultured in, for example, DMEM-LG (Dulbecco's ModifiedEssential Medium, low glucose)/MCDB 201 (chick fibroblast basal medium)containing ITS (insulin-transferrin-selenium), LA+BSA (linoleicacid-bovine serum albumin), dextrose, L-ascorbic acid, PDGF, EGF, IGF-1,and penicillin/streptomycin; DMEM-HG (high glucose) comprising 10% fetalbovine serum (FBS); DMEM-HG comprising 15% FBS; IMDM (Iscove's modifiedDulbecco's medium) comprising 10% FBS, 10% horse serum, andhydrocortisone; M199 comprising 10% FBS, EGF, and heparin; α-MEM(minimal essential medium) comprising 10% FBS, GLUTAMAX™ and gentamicin;DMEM comprising 10% FBS, GLUTAMAX™ and gentamicin, etc. A preferredmedium is DMEM-LG/MCDB-201 comprising 2% FBS, ITS, LA+BSA, dextrose,L-ascorbic acid, PDGF, EGF, and penicillin/streptomycin.

Other media in that can be used to culture placental stem cells includeDMEM (high or low glucose), Eagle's basal medium, Ham's F10 medium(F10), Ham's F-12 medium (F12), Iscove's modified Dulbecco's medium,Mesenchymal Stem Cell Growth Medium (MSCGM), Liebovitz's L-15 medium,MCDB, DMEM/F12, RPMI 1640, advanced DMEM (Gibco), DMEM/MCDB201 (Sigma),and CELL-GRO FREE.

The culture medium can be supplemented with one or more componentsincluding, for example, serum (e.g., fetal bovine serum (FBS),preferably about 2-15% (v/v); equine (horse) serum (ES); human serum(HS)); beta-mercaptoethanol (BME), preferably about 0.001% (v/v); one ormore growth factors, for example, platelet-derived growth factor (PDGF),epidermal growth factor (EGF), basic fibroblast growth factor (bFGF),insulin-like growth factor-1 (IGF-1), leukemia inhibitory factor (LIF),vascular endothelial growth factor (VEGF), and erythropoietin (EPO);amino acids, including L-valine; and one or more antibiotic and/orantimycotic agents to control microbial contamination, such as, forexample, penicillin G, streptomycin sulfate, amphotericin B, gentamicin,and nystatin, either alone or in combination.

Placental stem cells can be cultured in standard tissue cultureconditions, e.g., in tissue culture dishes or multiwell plates.Placental stem cells can also be cultured using a hanging drop method.In this method, placental stem cells are suspended at about 1×10⁴ cellsper mL in about 5 mL of medium, and one or more drops of the medium areplaced on the inside of the lid of a tissue culture container, e.g., a100 mL Petri dish. The drops can be, e.g., single drops, or multipledrops from, e.g., a multichannel pipetter. The lid is carefully invertedand placed on top of the bottom of the dish, which contains a volume ofliquid, e.g., sterile PBS sufficient to maintain the moisture content inthe dish atmosphere, and the stem cells are cultured.

5.3.2 Expansion and Proliferation of Placental Stem Cells

Once an isolated placental stem cell, or isolated population of stemcells (e.g., a stem cell or population of stem cells separated from atleast about 50% of the placental cells with which the stem cell orpopulation of stem cells is normally associated in vivo), the stem cellor population of stem cells can be proliferated and expanded in vitro.For example, a population of placental stem cells can be cultured intissue culture containers, e.g., dishes, flasks, multiwell plates, orthe like, for a sufficient time for the stem cells to proliferate to70-90% confluence, that is, until the stem cells and their progenyoccupy 70-90% of the culturing surface area of the tissue culturecontainer.

Placental stem cells can be seeded in culture vessels at a density thatallows cell growth. For example, the cells may be seeded at low density(e.g., about 1,000 to about 5,000 cells/cm²) to high density (e.g.,about 50,000 or more cells/cm²). In a preferred embodiment, the cellsare cultured at about 0 to about 5 percent by volume CO₂ in air. In somepreferred embodiments, the cells are cultured at about 2 to about 25percent O₂ in air, preferably about 5 to about 20 percent O₂ in air. Thecells preferably are cultured at about 25° C. to about 40° C.,preferably 37° C. The cells are preferably cultured in an incubator. Theculture medium can be static or agitated, for example, using abioreactor. Placental stem cells preferably are grown under lowoxidative stress (e.g., with addition of glutathione, ascorbic acid,catalase, tocopherol, N-acetylcysteine, or the like).

Once 70%-90% confluence is obtained, the cells may be passaged. Forexample, the cells can be enzymatically treated, e.g., trypsinized,using techniques well-known in the art, to separate them from the tissueculture surface. After removing the cells by pipetting and counting thecells, about 20,000-100,000 stem cells, preferably about 50,000 stemcells, are passaged to a new culture container containing fresh culturemedium. Typically, the new medium is the same type of medium from whichthe stem cells were removed. Provided herein are populations ofplacental stem cells that have been passaged at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 12, 14, 16, 18, or 20 times, or more.

5.3.3 Placental Stem Cell Populations

Further provided herein are populations of placental stem cells.Placental stem cell population can be isolated directly from one or moreplacentas; that is, the placental stem cell population can be apopulation of placental cells, comprising placental stem cells, obtainedfrom, or contained within, perfusate, or obtained from, or containedwithin, digestate (that is, the collection of cells obtained byenzymatic digestion of a placenta or part thereof). Isolated placentalstem cells provided herein can also be cultured and expanded to produceplacental stem cell populations. Populations of placental cellscomprising placental stem cells can also be cultured and expanded toproduce placental stem cell populations.

Placental stem cell populations provided herein comprise placental stemcells, for example, placental stem cells as described herein. In variousembodiments, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, or 99% of the cells in an isolated placental stem cell populationare placental stem cells. That is, a placental stem cell population cancomprise, e.g., as much as 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% non-stem cells.

Provided herein are methods of producing isolated placental stem cellpopulation by, e.g., selecting placental stem cells, whether derivedfrom enzymatic digestion or perfusion, that express particular markersand/or particular culture or morphological characteristics. In oneembodiment, for example, provided herein is a method of producing a cellpopulation comprising selecting placental cells that (a) adhere to asubstrate, and (b) express CD200 and HLA-G; and isolating said cellsfrom other cells to form a cell population. In another embodiment, themethod of producing a cell population comprises selecting placentalcells that (a) adhere to a substrate, and (b) express CD73, CD105, andCD200; and isolating said cells from other cells to form a cellpopulation. In another embodiment, the method of producing a cellpopulation comprises selecting placental cells that (a) adhere to asubstrate and (b) express CD200 and OCT-4; and isolating said cells fromother cells to form a cell population. In another embodiment, the methodof producing a cell population comprises selecting placental cells that(a) adhere to a substrate, (b) express CD73 and CD105, and (c)facilitate the formation of one or more embryoid-like bodies in apopulation of placental cells comprising said stem cell when saidpopulation is cultured under conditions that allow for the formation ofan embryoid-like body; and isolating said cells from other cells to forma cell population. In another embodiment, the method of producing a cellpopulation comprises selecting placental cells that (a) adhere to asubstrate, and (b) express CD73, CD105 and HLA-G; and isolating saidcells from other cells to form a cell population. In another embodiment,the method of producing a cell population comprises selecting placentalcells that (a) adhere to a substrate, (b) express OCT-4, and (c)facilitate the formation of one or more embryoid-like bodies in apopulation of placental cells comprising said stem cell when saidpopulation is cultured under conditions that allow for the formation ofan embryoid-like body; and isolating said cells from other cells to forma cell population. In any of the above embodiments, the method canadditionally comprise selecting placental cells that express ABC-p (aplacenta-specific ABC transporter protein; see, e.g., Allikmets et al.,Cancer Res. 58(23):5337-9 (1998)). The method can also compriseselecting cells exhibiting at least one characteristic specific to,e.g., a mesenchymal stem cell, for example, expression of CD29,expression of CD44, expression of CD90, or expression of a combinationof the foregoing.

In the above embodiments, the substrate can be any surface on whichculture and/or selection of cells, e.g., placental stem cells, can beaccomplished. Typically, the substrate is plastic, e.g., tissue culturedish or multiwell plate plastic. Tissue culture plastic can be coatedwith a biomolecule, e.g., laminin or fibronectin.

Cells, e.g., placental stem cells, can be selected for a placental stemcell population by any means known in the art of cell selection. Forexample, cells can be selected using an antibody or antibodies to one ormore cell surface markers, for example, in flow cytometry or FACS.Selection can be accomplished using antibodies in conjunction withmagnetic beads. Antibodies that are specific for certain stemcell-related markers are known in the art. For example, antibodies toOCT-4 (Abcam, Cambridge, Mass.), CD200 (Abcam), HLA-G (Abcam), CD73 (BDBiosciences Pharmingen, San Diego, Calif.), CD105 (Abcam; BioDesignInternational, Saco, Me.), etc. Antibodies to other markers are alsoavailable commercially, e.g., CD34, CD38 and CD45 are available from,e.g., StemCell Technologies or BioDesign International.

The isolated placental stem cell population can comprise placental cellsthat are not stem cells, or cells that are not placental cells.

Isolated placental stem cell populations can be combined with one ormore populations of non-stem cells or non-placental cells. For example,an isolated population of placental stem cells can be combined withblood (e.g., placental blood or umbilical cord blood), blood-derivedstem cells (e.g., stem cells derived from placental blood or umbilicalcord blood), populations of blood-derived nucleated cells, bonemarrow-derived mesenchymal cells, bone-derived stem cell populations,crude bone marrow, adult (somatic) stem cells, populations of stem cellscontained within tissue, cultured stem cells, populations offully-differentiated cells (e.g., chondrocytes, fibroblasts, amnioticcells, osteoblasts, muscle cells, cardiac cells, etc.) and the like.Cells in an isolated placental stem cell population can be combined witha plurality of cells of another type in ratios of about 100,000,000:1,50,000,000:1, 20,000,000:1, 10,000,000:1, 5,000,000:1, 2,000,000:1,1,000,000:1, 500,000:1, 200,000:1, 100,000:1, 50,000:1, 20,000:1,10,000:1, 5,000:1, 2,000:1, 1,000:1, 500:1, 200:1, 100:1, 50:1, 20:1,10:1, 5:1, 2:1, 1:1; 1:2; 1:5; 1:10; 1:100; 1:200; 1:500; 1:1,000;1:2,000; 1:5,000; 1:10,000; 1:20,000; 1:50,000; 1:100,000; 1:500,000;1:1,000,000; 1:2,000,000; 1:5,000,000; 1:10,000,000; 1:20,000,000;1:50,000,000; or about 1:100,000,000, comparing numbers of totalnucleated cells in each population. Cells in an isolated placental stemcell population can be combined with a plurality of cells of a pluralityof cell types, as well.

In one, an isolated population of placental stem cells is combined witha plurality of hematopoietic stem cells. Such hematopoietic stem cellscan be, for example, contained within unprocessed placental, umbilicalcord blood or peripheral blood; in total nucleated cells from placentalblood, umbilical cord blood or peripheral blood; in an isolatedpopulation of CD34⁺ cells from placental blood, umbilical cord blood orperipheral blood; in unprocessed bone marrow; in total nucleated cellsfrom bone marrow; in an isolated population of CD34⁺ cells from bonemarrow, or the like.

5.4 Combinations of Placental Stem Cells and Placental Perfusate orPlacental Perfusate Cells

Provided herein are combinations of placental perfusate with isolatedplacental perfusate cells and/or the placental stem cells provided.Herein, the placental stem cells can be CD34⁺ placental stem cells,CD34⁻ placental stem cells, or a combination thereof. In one embodiment,for example, provided herein is a volume of placental perfusatesupplemented with a plurality of placental perfusate cells and/or aplurality of placental stem cells. In specific embodiments, for example,each milliliter of placental perfusate is supplemented with about 1×10⁴,5×10⁴, 1×10⁵, 5×10⁵, 1×10⁶, 5×10⁶ or more placental perfusate cells orplacental stem cells. In another embodiment, a plurality of placentalperfusate cells is supplemented with placental perfusate and/orplacental stem cells. In another embodiment, a plurality of placentalstem cells is supplemented with placental perfusate and/or a pluralityof placental perfusate cells. In certain embodiments, when perfusate isused for supplementation, the volume of perfusate is about, greater thanabout, or less than about, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%,8%, 6%, 4%, 2% or 1% of the total volume of cells (in solution) plusperfusate. When placental perfusate cells are used to supplement aplurality of placental stem cells, the placental perfusate cellsgenerally comprise about, greater than about, or fewer than about, 50%,45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 8%, 6%, 4%, 2% or 1% of thetotal number of placental perfusate cells plus placental stem cells.Similarly, when placental stem cells are used to supplement a pluralityof placental perfusate cells, the placental stem cells generallycomprise about, greater than about, or fewer than about, 50%, 45%, 40%,35%, 30%, 25%, 20%, 15%, 10%, 8%, 6%, 4%, 2% or 1% of the total numberof placental perfusate cells plus placental stem cells. When placentalstem cells or placental perfusate cells are used to supplement placentalperfusate, the volume of solution (e.g., saline solution, culture mediumor the like) in which the cells are suspended comprises about, greaterthan about, or less than about, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%,10%, 8%, 6%, 4%, 2% or 1% of the total volume of perfusate plus cells,where the placental stem cells are suspended to about 1×10⁴, 5×10⁴,1×10⁵, 5×10⁵, 1×10⁶, 5×10⁶, 1×10⁷, 5×10⁷, 1×10⁸, 5×10⁸ or more cells permilliliter prior to supplementation.

Further provided herein is pooled placental perfusate that is obtainedfrom two or more sources, e.g., two or more placentas, and combined,e.g., pooled. Such pooled perfusate can comprise approximately equalvolumes of perfusate from each source, or can comprise different volumesfrom each source. The relative volumes from each source can be randomlyselected, or can be based upon, e.g., a concentration or amount of oneor more cellular factors, e.g., cytokines, growth factors, hormones, orthe like; the number of placental cells in perfusate from each source;or other characteristics of the perfusate from each source. Perfusatefrom multiple perfusions of the same placenta can similarly be pooled.

Similarly, provided herein are placental perfusate cells, and placentalstem cells, that are obtained from two or more sources, e.g., two ormore placentas, and pooled. Such pooled cells can comprise approximatelyequal numbers of cells from the two or more sources, or differentnumbers of cells from one or more of the pooled sources. The relativenumbers of cells from each source can be selected based on, e.g., thenumber of one or more specific cell types in the cells to be pooled,e.g., the number of CD34⁻ stem cells, etc.

Pools can comprise, e.g., placental perfusate supplemented withplacental perfusate cells; placental perfusate supplemented withplacental stem cells; placental perfusate supplemented with bothplacental perfusate cells and placental stem cells; placental perfusatecells supplemented with placental perfusate; placental perfusate cellssupplemented with placental stem cells; placental perfusate cellssupplemented with both placental perfusate and placental stem cells;placental stem cells supplemented with placental perfusate; placentalstem cells supplemented with placental perfusate cells; or placentalstem cells supplemented with both placental perfusate cells andplacental perfusate.

In certain embodiments, placental perfusate, placental perfusate cells,and placental stem cells are provided as pharmaceutical gradeadministrable units. Such units can be provided in discrete volumes,e.g., 100 mL, 150 mL, 200 mL, 250 mL, 300 mL, 350 mL, 400 mL, 450 mL,500 mL, or the like. Such units can be provided so as to contain aspecified number of, e.g., placental perfusate cells, placentalperfusate-derived intermediate natural killer cells, or both, e.g.,1×10⁴, 5×10⁴, 1×10⁵, 5×10⁵, 1×10⁶, 5×10⁶, 1×10⁷, 5×10⁷, 1×10⁸, 5×10⁸ ormore cells per milliliter, or 1×10⁴, 5×10⁴, 1×10⁵, 5×10⁵, 1×10⁶, 5×10⁶,1×10⁷, 5×10⁷, 1×10⁸, 5×10⁸, 1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰, 1×10¹¹ or morecells per unit. Such units can be provided to contain specified numbersof any two, or all three, of placental perfusate, placental perfusatecells, and/or placental stem cells.

In the above combinations of placental perfusate, placental perfusatecells and/or placental stem cells, any one, any two, or all three of theplacental perfusate, placental perfusate cells and/or placental stemcells can be autologous to a recipient (that is, obtained from therecipient), or homologous to a recipient (that is, obtained from at lastone other individual from said recipient).

Also provided herein are compositions comprising placental stem cells incombination with placental perfusate cells and/or placental perfusate.Thus, in another aspect, provided herein is a composition comprisingisolated placental stem cells, wherein said placental stem are isolatedfrom placental perfusate, and wherein said placental stem cells compriseat least 50% of cells in the composition. In a specific embodiment, saidplacental stem cells comprise at least 80% of cells in the composition.In another specific embodiment, the composition comprises isolatedplacental perfusate. In a more specific embodiment, said placentalperfusate is from the same individual as said placental stem cells. Inanother more specific embodiment, said placental perfusate comprisesplacental perfusate from a different individual than said placental stemcells. In another specific embodiment, the composition comprisesplacental perfusate cells. In a more specific embodiment, said placentalperfusate cells are from the same individual as said placental stemcells. In another more specific embodiment, said placental perfusatecells are from a different individual than said placental stem cells. Inanother specific embodiment, the composition additionally comprisesisolated placental perfusate and isolated placental perfusate cells,wherein said isolated perfusate and said isolated placental perfusatecells are from different individuals. In another more specificembodiment of any of the above embodiments comprising placentalperfusate, said placental perfusate comprises placental perfusate fromat least two individuals. In another more specific embodiment of any ofthe above embodiments comprising placental perfusate cells, saidisolated placental perfusate cells are from at least two individuals.

5.5 Production of a Placental Stem Cell Bank

Stem cells from postpartum placentas can be cultured in a number ofdifferent ways to produce a set of lots, e.g., a set ofindividually-administrable doses, of placental stem cells. Such lotscan, for example, be obtained from stem cells from placental perfusateor from enzyme-digested placental tissue. Sets of lots of placental stemcells, obtained from a plurality of placentas, can be arranged in a bankof placental stem cells for, e.g., long-term storage. Generally,adherent stem cells are obtained from an initial culture of placentalmaterial to form a seed culture, which is expanded under controlledconditions to form populations of cells from approximately equivalentnumbers of doublings. Lots are preferably derived from the tissue of asingle placenta, but can be derived from the tissue of a plurality ofplacentas.

In one embodiment, stem cell lots are obtained as follows. Placentaltissue is first disrupted, e.g., by mincing, digested with a suitableenzyme, e.g., collagenase (see Section 5.2.3, above). The placentaltissue preferably comprises, e.g., the entire amnion, entire chorion, orboth, from a single placenta, but can comprise only a part of either theamnion or chorion. The digested tissue is cultured, e.g., for about 1-3weeks, preferably about 2 weeks. After removal of non-adherent cells,high-density colonies that form are collected, e.g., by trypsinization.These cells are collected and resuspended in a convenient volume ofculture medium, and defined as Passage 0 cells.

Passage 0 cells are then used to seed expansion cultures. Expansioncultures can be any arrangement of separate cell culture apparatuses,e.g., a Cell Factory by NUNC™. Cells in the Passage 0 culture can besubdivided to any degree so as to seed expansion cultures with, e.g.,1×10³, 2×10³, 3×10³, 4×10³, 5×10³, 6×10³, 7×10³, 8×10³, 9×10³, 1×10⁴,1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10, or 10×10⁴stem cells. Preferably, from about 2×10⁴ to about 3×10⁴ Passage 0 cellsare used to seed each expansion culture. The number of expansioncultures can depend upon the number of Passage 0 cells, and may begreater or fewer in number depending upon the particular placenta(s)from which the stem cells are obtained.

Expansion cultures are grown until the density of cells in culturereaches a certain value, e.g., about 1×10⁵ cells/cm². Cells can eitherbe collected and cryopreserved at this point, or passaged into newexpansion cultures as described above. Cells can be passaged, e.g., 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 timesprior to use. A record of the cumulative number of population doublingsis preferably maintained during expansion culture(s). The cells from aPassage 0 culture can be expanded for 2, 3, 4, 5, 6, 7, 8, 9, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or 40 doublings, orup to 60 doublings. Preferably, however, the number of populationdoublings, prior to dividing the population of cells into individualdoses, is between about 15 and about 30, preferably about 20 doublings.The cells can be culture continuously throughout the expansion process,or can be frozen at one or more points during expansion.

Cells to be used for individual doses can be frozen, e.g., cryopreservedfor later use. Individual doses can comprise, e.g., about 1 million toabout 100 million cells per ml, and can comprise between about 10⁶ andabout 10⁹ cells in total.

In a specific embodiment, of the method, Passage 0 cells are culturedfor approximately 4 doublings, then frozen in a first cell bank. Cellsfrom the first cell bank are frozen and used to seed a second cell bank,the cells of which are expanded for about another eight doublings. Cellsat this stage are collected and frozen and used to seed new expansioncultures that are allowed to proceed for about eight additionaldoublings, bringing the cumulative number of cell doublings to about 20.Cells at the intermediate points in passaging can be frozen in units ofabout 100,000 to about 10 million cells per ml, preferably about 1million cells per ml for use in subsequent expansion culture. Cells atabout 20 doublings can be frozen in individual doses of between about 1million to about 100 million cells per ml for administration or use inmaking a stem cell-containing composition.

In a preferred embodiment, the donor from which the placenta is obtained(e.g., the mother) is tested for at least one pathogen. If the mothertests positive for a tested pathogen, the entire lot from the placentais discarded. Such testing can be performed at any time duringproduction of placental stem cell lots, including before or afterestablishment of Passage 0 cells, or during expansion culture. Pathogensfor which the presence is tested can include, without limitation,hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, humanimmunodeficiency virus (types I and II), cytomegalovirus, herpesvirus,and the like.

5.6 Differentiation of Adherent Placental Stem Cells 5.6.1 Induction ofDifferentiation into Neuronal or Neurogenic Cells

Neuronal differentiation of placental stem cells can be accomplished,for example, by placing placental stem cells in cell culture conditionsthat induce differentiation into neurons. In an example method, aneurogenic medium comprises DMEM/20% FBS and 1 mM beta-mercaptoethanol;such medium can be replaced after culture for about 24 hours with mediumconsisting of DMEM and 1-10 mM betamercaptoethanol. In anotherembodiment, the cells are contacted with DMEM/2% DMSO/200 μM butylatedhydroxyanisole. In a specific embodiment, the differentiation mediumcomprises serum-free DMEM F-12, butylated hydroxyanisole, potassiumchloride, insulin, forskolin, valproic acid, and hydrocortisone. Inanother embodiment, neuronal differentiation is accomplished by platingplacental stem cells on laminin-coated plates in Neurobasal-A medium(Invitrogen, Carlsbad Calif.) containing B27 supplement and L-glutamine,optionally supplemented with bFGF and/or EGF. Placental stem cells canalso be induced to neural differentiation by co-culture with neuralcells, or culture in neuron-conditioned medium.

Neuronal differentiation can be assessed, e.g., by detection ofneuron-like morphology (e.g., bipolar cells comprising extendedprocesses) detection of the expression of e.g., nerve growth factorreceptor and neurofilament heavy chain genes by RT/PCR; or detection ofelectrical activity, e.g., by patch-clamp.

5.6.2 Induction of Differentiation into Adipogenic Cells

Adipogenic differentiation of placental stem cells can be accomplished,for example, by placing placental stem cells in cell culture conditionsthat induce differentiation into adipocytes. A preferred adipogenicmedium comprises MSCGM (Cambrex) or DMEM supplemented with 15% cordblood serum. In one embodiment, placental stem cells are fedAdipogenesis Induction Medium (Cambrex) and cultured for 3 days (at 37°C., 5% CO₂), followed by 1-3 days of culture in Adipogenesis MaintenanceMedium (Cambrex). After 3 complete cycles of induction/maintenance, thecells are cultured for an additional 7 days in adipogenesis maintenancemedium, replacing the medium every 2-3 days.

In another embodiment, placental stem cells are cultured in mediumcomprising 1 μM dexamethasone, 0.2 mM indomethacin, 0.01 mg/ml insulin,0.5 mM IBMX, DMEM-high glucose, FBS, and antibiotics. Placental stemcells can also be induced towards adipogenesis by culture in mediumcomprising one or more glucocorticoids (e.g., dexamethasone,indomethasone, hydrocortisone, cortisone), insulin, a compound whichelevates intracellular levels of cAMP (e.g., dibutyryl-cAMP; 8-CPT-cAMP(8-(4)chlorophenylthio)-adenosine, 3′,5′ cyclic monophosphate);8-bromo-cAMP; dioctanoyl-cAMP; forskolin) and/or a compound whichinhibits degradation of cAMP (e.g., a phosphodiesterase inhibitor suchas isobutylmethylxanthine (IBMX), methyl isobutylxanthine, theophylline,caffeine, indomethacin).

A hallmark of adipogenesis is the development of multipleintracytoplasmic lipid vesicles that can be easily observed using thelipophilic stain oil red 0. Expression of lipase and/or fatty acidbinding protein genes is confirmed by RT/PCR in placental stem cellsthat have begun to differentiate into adipocytes.

5.6.3 Induction of Differentiation into Chondrocytic Cells

Chondrogenic differentiation of placental stem cells can beaccomplished, for example, by placing placental stem cells in cellculture conditions that induce differentiation into chondrocytes. Apreferred chondrocytic medium comprises MSCGM (Cambrex) or DMEMsupplemented with 15% cord blood serum. In one embodiment, placentalstem cells are aliquoted into a sterile polypropylene tube, centrifuged(e.g., at 150×g for 5 minutes), and washed twice in IncompleteChondrogenesis Medium (Cambrex). The cells are resuspended in CompleteChondrogenesis Medium (Cambrex) containing 0.01 μg/ml TGF-beta-3 at aconcentration of about 1-20×10⁵ cells/ml. In other embodiments,placental stem cells are contacted with exogenous growth factors, e.g.,GDF-5 or transforming growth factor beta3 (TGF-beta3), with or withoutascorbate. Chondrogenic medium can be supplemented with amino acidsincluding proline and glutamine, sodium pyruvate, dexamethasone,ascorbic acid, and insulin/transferrin/selenium. Chondrogenic medium canbe supplemented with sodium hydroxide and/or collagen. The placentalstem cells may be cultured at high or low density. Cells are preferablycultured in the absence of serum.

Chondrogenesis can be assessed by e.g., observation of production ofesoinophilic ground substance, safranin-O staining for glycosaminoglycanexpression; hematoxylin/eosin staining, assessing cell morphology,and/or RT/PCR confirmation of collagen 2 and collagen 9 gene expression.Chondrogenesis can also be observed by growing the stem cells in apellet, formed, e.g., by gently centrifuging stem cells in suspension(e.g., at about 800 g for about 5 minutes). After about 1-28 days, thepellet of stem cells begins to form a tough matrix and demonstrates astructural integrity not found in non-induced, or non-chondrogenic, celllines, pellets of which tend to fall apart when challenged.Chondrogenesis can also be demonstrated, e.g., in such cell pellets, bystaining with a stain that stains collage, e.g., Sirius Red, and/or astain that stains glycosaminoglycans (GAGs), such as, e.g., Alcian Blue.

5.6.4 Induction of Differentiation into Osteogenic Cells

Osteogenic differentiation of placental stem cells can be accomplished,for example, by placing placental stem cells in cell culture conditionsthat induce differentiation into osteogenic cells. A preferredosteocytic medium comprises MSCGM (Cambrex) or DMEM supplemented with15% cord blood serum, followed by Osteogenic Induction Medium (Cambrex)containing 0.1 μM dexamethasone, 0.05 mM ascorbic acid-2-phosphate, 10mM beta glycerophosphate. In another embodiment, placental stem cellsare cultured in medium (e.g., DMEM-low glucose) containing about 10⁻⁷ toabout 10⁻⁹ M dexamethasone, about 10-50 μM ascorbate phosphate salt(e.g., ascorbate-2-phosphate) and about 10 nM to about 10 mMβ-glycerophosphate. Osteogenic medium can also include serum, one ormore antibiotic/antimycotic agents, transforming growth factor-beta(e.g., TGF-β1) and/or bone morphogenic protein (e.g., BMP-2, BMP-4, or acombination thereof).

Differentiation can be assayed using a calcium-specific stain, e.g., vonKossa staining, and RT/PCR detection of, e.g., alkaline phosphatase,osteocalcin, bone sialoprotein and/or osteopontin gene expression.

5.6.5 Induction of Differentiation into Pancreatic Cells

Differentiation of placental stem cells into insulin-producingpancreatic cells can be accomplished, for example, by placing placentalstem cells in cell culture conditions that induce differentiation intopancreatic cells.

An example pancreagenic medium comprises DMEM/20% CBS, supplemented withbasic fibroblast growth factor, 10 ng/ml; and transforming growth factorbeta-1, 2 ng/ml. This medium is combined with conditioned media fromnestin-positive neuronal cell cultures at 50/50 v/v. KnockOut SerumReplacement can be used in lieu of CBS. Cells are cultured for 14-28days, refeeding every 3-4 days.

Differentiation can be confirmed by assaying for, e.g., insulin proteinproduction, or insulin gene expression by RT/PCR.

5.6.6 Induction of Differentiation into Cardiac Cells

Myogenic (cardiogenic) differentiation of placental stem cells can beaccomplished, for example, by placing placental stem cells in cellculture conditions that induce differentiation into cardiomyocytes. Apreferred cardiomyocytic medium comprises DMEM/20% CBS supplemented withretinoic acid, 1 μM; basic fibroblast growth factor, 10 ng/ml; andtransforming growth factor beta-1, 2 ng/ml; and epidermal growth factor,100 ng/ml. KnockOut Serum Replacement (Invitrogen, Carlsbad, Calif.) maybe used in lieu of CBS. Alternatively, placental stem cells are culturedin DMEM/20% CBS supplemented with 50 ng/ml Cardiotropin-1 for 24 hours.In another embodiment, placental stem cells can be cultured 10-14 daysin protein-free medium for 5-7 days, then stimulated with humanmyocardium extract, e.g., produced by homogenizing human myocardium in1% HEPES buffer supplemented with 1% cord blood serum.

Differentiation can be confirmed by demonstration of cardiac actin geneexpression, e.g., by RT/PCR.

5.7 Preservation of Placental Stem Cells

Placental stem cells can be preserved, that is, placed under conditionsthat allow for long-term storage, or conditions that inhibit cell deathby, e.g., apoptosis or necrosis.

Placental stem cells can be preserved using, e.g., a compositioncomprising an apoptosis inhibitor, necrosis inhibitor and/or anoxygen-carrying perfluorocarbon, as described in related U.S.Provisional Application No. 60/754,969, entitled “Improved Medium forCollecting Placental Stem Cells and Preserving Organs,” filed on Dec.25, 2005. In one embodiment, provided herein is a method of preserving apopulation of stem cells comprising contacting said population of stemcells with a stem cell collection composition comprising an inhibitor ofapoptosis and an oxygen-carrying perfluorocarbon, wherein said inhibitorof apoptosis is present in an amount and for a time sufficient to reduceor prevent apoptosis in the population of stem cells, as compared to apopulation of stem cells not contacted with the inhibitor of apoptosis.In a specific embodiment, said inhibitor of apoptosis is a caspaseinhibitor. In another specific embodiment, said inhibitor of apoptosisis a JNK inhibitor. In a more specific embodiment, said JNK inhibitordoes not modulate differentiation or proliferation of said stem cells.In another embodiment, said stem cell collection composition comprisessaid inhibitor of apoptosis and said oxygen-carrying perfluorocarbon inseparate phases. In another embodiment, said stem cell collectioncomposition comprises said inhibitor of apoptosis and saidoxygen-carrying perfluorocarbon in an emulsion. In another embodiment,the stem cell collection composition additionally comprises anemulsifier, e.g., lecithin. In another embodiment, said apoptosisinhibitor and said perfluorocarbon are between about 0° C. and about 25°C. at the time of contacting the stem cells. In another more specificembodiment, said apoptosis inhibitor and said perfluorocarbon arebetween about 2° C. and 10° C., or between about 2° C. and about 5° C.,at the time of contacting the stem cells. In another more specificembodiment, said contacting is performed during transport of saidpopulation of stem cells. In another more specific embodiment, saidcontacting is performed during freezing and thawing of said populationof stem cells.

In another embodiment, provided herein is a method of preserving apopulation of placental stem cells comprising contacting said populationof stem cells with an inhibitor of apoptosis and an organ-preservingcompound, wherein said inhibitor of apoptosis is present in an amountand for a time sufficient to reduce or prevent apoptosis in thepopulation of stem cells, as compared to a population of stem cells notcontacted with the inhibitor of apoptosis. In a specific embodiment, theorgan-preserving compound is UW solution (described in U.S. Pat. No.4,798,824; also known as ViaSpan; see also Southard et al.,Transplantation 49(2):251-257 (1990)) or a solution described in Sternet al., U.S. Pat. No. 5,552,267. In another embodiment, saidorgan-preserving compound is hydroxyethyl starch, lactobionic acid,raffinose, or a combination thereof. In another embodiment, the stemcell collection composition additionally comprises an oxygen-carryingperfluorocarbon, either in two phases or as an emulsion.

In another embodiment of the method, placental stem cells are contactedwith a stem cell collection composition comprising an apoptosisinhibitor and oxygen-carrying perfluorocarbon, organ-preservingcompound, or combination thereof, during perfusion. In anotherembodiment, said stem cells are contacted during a process of tissuedisruption, e.g., enzymatic digestion. In another embodiment, placentalstem cells are contacted with said stem cell collection compound aftercollection by perfusion, or after collection by tissue disruption, e.g.,enzymatic digestion.

Typically, during placental cell collection, enrichment and isolation,it is preferable to minimize or eliminate cell stress due to hypoxia andmechanical stress. In another embodiment of the method, therefore, astem cell, or population of stem cells, is exposed to a hypoxiccondition during collection, enrichment or isolation for less than sixhours during said preservation, wherein a hypoxic condition is aconcentration of oxygen that is less than normal blood oxygenconcentration. In a more specific embodiment, said population of stemcells is exposed to said hypoxic condition for less than two hoursduring said preservation. In another more specific embodiment, saidpopulation of stem cells is exposed to said hypoxic condition for lessthan one hour, or less than thirty minutes, or is not exposed to ahypoxic condition, during collection, enrichment or isolation. Inanother specific embodiment, said population of stem cells is notexposed to shear stress during collection, enrichment or isolation.

The placental stem cells provided herein can be cryopreserved, e.g., incryopreservation medium in small containers, e.g., ampoules. Suitablecryopreservation medium includes, but is not limited to, culture mediumincluding, e.g., growth medium, or cell freezing medium, for examplecommercially available cell freezing medium, e.g., C2695, C2639 or C6039(Sigma). Cryopreservation medium preferably comprises DMSO(dimethylsulfoxide), at a concentration of, e.g., about 10% (v/v).Cryopreservation medium may comprise additional agents, for example,methylcellulose and/or glycerol. Placental stem cells are preferablycooled at about 1° C./min during cryopreservation. A preferredcryopreservation temperature is about −80° C. to about −180° C.,preferably about −125° C. to about −140° C. Cryopreserved cells can betransferred to liquid nitrogen prior to thawing for use. In someembodiments, for example, once the ampoules have reached about −90° C.,they are transferred to a liquid nitrogen storage area. Cryopreservedcells preferably are thawed at a temperature of about 25° C. to about40° C., preferably to a temperature of about 37° C.

5.8 Uses of Placental Stem Cells 5.8.1 Placental Perfusate, Stem Cellsand Stem Cell Populations

Placental stem cell populations can be used to treat any disease,disorder or condition that is amenable to treatment by administration ofa population of stem cells. As used herein, “treat” encompasses the cureof, remediation of, improvement of, lessening of the severity of, orreduction in the time course of, a disease, disorder or condition, orany parameter or symptom thereof.

Placental stem cells, and populations of placental stem cells, can beinduced to differentiate into a particular cell type, either ex vivo orin vivo, in preparation for administration to an individual in need ofstem cells, or cells differentiated from stem cells. For example,placental stem cells can be injected into a damaged organ, and for organneogenesis and repair of injury in vivo. Such injury may be due to suchconditions and disorders including, but not limited to, bone defectsincluding lesions resulting from cancer, fractures, and spinalconditions treatable with, e.g., spinal fusion. The placental stem cellscan be injected into the damaged bone alone or can be introduced with animplantable substrate as described herein. Isolated populations ofplacental stem cells can be used, in specific embodiments, to treatspecific diseases or conditions, including, but not limited to multiplemyeloma, cancers including bone cancer, neuroblastoma, osteosarcoma,Ewing's sarcoma, chondrosarcoma, chordoma, malignant fibroushistiocytoma of bone, fibrosarcoma of bone, metastatic cancer, multiplemyeloma, and any form of metastatic cancer characterized by bonemetastases. As one skilled in the art will recognize, treatment of bonedefects caused by cancer will not necessarily abate the cancer itself.Treatment of bone defects as provided herein can occur before, after, orconcurrently with additional cancer therapies. Accordingly, in oneembodiment, bone defects are treated before the cancer is treated withan anti-cancer therapy. In another embodiment, bone defects are treatedat or near the same time that the cancer is treated with an anti-cancertherapy. In another embodiment, bone defects are treated after thecancer is treated with an anti-cancer therapy.

Isolated placental perfusate, placental perfusate cells, and/or isolatedpopulations of placental stem cells may also be used to treat bonefractures, e.g., non-union bone fractures. Isolated populations ofplacental stem cells may also be used to fuse vertebrae together inorder to, e.g., complete a spinal fusion in a subject in need thereof.Isolated populations of placental stem cells, in combination with stemor progenitor cell populations, may also be used to treat the foregoing.

In certain embodiments of the above methods of treating bone defects,placental perfusate, placental perfusate cells and/or placental stemcells, e.g., adherent or nonadherent placental stem cells, can beadministered to an individual having a bone defect. Such an individualcan be administered with, e.g., placental perfusate as obtained from aplacenta; placental perfusate that has been treated to remove one ormore cell types, e.g., erythrocytes; placental perfusate cells isolatedfrom placental perfusate, or combinations of any of the foregoing. Suchcombinations can also comprise isolated adherent placental stem cellsand or isolated nonadherent placental stem cells, as described elsewhereherein. Combinations of placental perfusate, isolated placentalperfusate cells and/or placental stem cells useful to treat a bonedefect, or an individual having a bone defect, are described in Section5.4, above.

In specific embodiments of the method of treatment, the placental cellsare contained within whole (unprocessed) placental perfusate. In anotherspecific embodiment, the placental cells are placental perfusate cells.In another specific embodiment, the placental cells are placental stemcells. In certain more specific embodiments, the stem cells arenonadherent. In certain embodiments, the stem cells are CD34⁺. Incertain embodiments, the stem cells are CD44⁻. In certain embodiments,the said stem cells are CD34⁺ and CD44⁻. In certain embodiments, thesaid stem cells are CD9⁺, CD54⁺, CD90⁺, or CD166⁺. In certainembodiments, the said stem cells are CD9⁺, CD54⁺, CD90⁺, and CD166⁺. Incertain embodiments, the said stem cells are CD31⁺, CD117⁺, CD133⁺, orCD200⁺. In certain embodiments, the said stem cells are CD31⁺, CD117⁺,CD133⁺, and CD200⁺. In certain embodiments, at least about 70% of saidcells are CD34⁺ and CD44⁻ stem cells. In certain embodiments, the atleast about 90% of said cells are CD34⁺ and CD44⁻ stem cells. In certainother embodiments of the method, the placental stem cells are adherent.In specific embodiments, the adherent placental stem cells are CD200⁺and HLA-G⁺; CD73⁺, CD105⁺, and CD200⁺; CD200⁺ and OCT-4⁺; CD73⁺, CD105⁺and HLA-G⁺; CD73⁺ and CD105⁺ and facilitates the formation of one ormore embryoid-like bodies in a population of placental cells comprisingsaid stem cell when said population is cultured under conditions thatallow the formation of an embryoid-like body; or OCT-4⁺ and facilitatesthe formation of one or more embryoid-like bodies in a population ofplacental cells comprising the stem cell when said population iscultured under conditions that allow formation of embryoid-like bodies;or any combination thereof. In more specific embodiments of thenonadherent placental stem cells, the isolated CD200⁺, HLA-G⁺ stem cellis CD34⁻, CD38⁻, CD45⁻, CD73⁺ and CD105⁺; the isolated CD73⁺, CD105⁺,and CD200⁺ stem cell is CD34⁻, CD38⁻, CD45⁻, and HLA-G⁺; the isolatedCD200⁺, OCT-4⁺ stem cell is CD34⁻, CD38⁻, CD45⁻, CD73⁺, CD105⁺ andHLA-G⁺; the isolated stem cell of claim 1, wherein said CD73⁺, CD105⁺and HLA-G⁺ stem cell is CD34⁻, CD45⁻, OCT-4⁺ and CD200⁺; the isolatedCD73⁺ and CD105⁺ stem cell that facilitates the formation of one or moreembryoid-like bodies is OCT4⁺, CD34⁻, CD38⁻ and CD45⁻; and/or theisolated OCT-4⁺ and which facilitates the formation of one or moreembryoid-like bodies is CD73⁺, CD105⁺, CD200⁺, CD34⁻, CD38⁻, and CD45⁻.In certain embodiments, the population of placental stem cells has beenexpanded.

When placental perfusate, placental perfusate cells, or placental stemcells are administered as a suspension or liquid injectable, the cellscan be administered intravenously, or, preferably, at the site of thebone defect, e.g., break.

Also provided herein is a method for treating bone defects in a subject,comprising administering to a subject in need thereof an implantable orinjectable composition comprising a population of stem cells providedherein, thereby treating the bone defect in the subject. In certainembodiments, the bone defect is an osteolytic lesion associated with acancer, a bone fracture, or a spine, e.g., in need of fusion. In certainembodiments, the osteolytic lesion is associated with multiple myeloma,bone cancer, or metastatic cancer. In certain embodiments, the bonefracture is a non-union fracture. In certain embodiments, an implantablecomposition comprising a population of nonadherent stem cells isadministered to the subject. In certain embodiments, an implantablecomposition is surgically implanted, e.g., at the site of the bonedefect. In certain embodiments, an injectable composition comprising apopulation of nonadherent stem cells is administered to the subject. Incertain embodiments, an injectable composition is surgicallyadministered to the region of the bone defect. In certain embodiments,the injectable composition is systemically administered.

In another aspect, provided herein is a method for formulating aninjectable composition, comprising combining a population of placentalcells with injectable hyaluronic acid or collagen. In a specificembodiment, the placental cells are contained within whole (unprocessed)placental perfusate. In another specific embodiment, the placental cellsare placental perfusate cells. In another specific embodiment, theplacental cells are placental stem cells. In certain more specificembodiments, the stem cells are nonadherent. In certain embodiments, thestem cells are CD34⁺. In certain embodiments, the stem cells are CD44⁻.In certain embodiments, the said stem cells are CD34⁺ and CD44⁻. Incertain embodiments, the said stem cells are CD9⁺, CD54⁺, CD90⁺, orCD166⁺. In certain embodiments, the said stem cells are CD9⁺, CD54⁺,CD90⁺, and CD166⁺. In certain embodiments, the said stem cells areCD31⁺, CD117⁺, CD133⁺, or CD200⁺. In certain embodiments, the said stemcells are CD31⁺, CD117⁺, CD133⁺, and CD200⁺. In certain embodiments, atleast about 70% of said cells are CD34⁺ and CD44⁻ stem cells. In certainembodiments, the at least about 90% of said cells are CD34⁺ and CD44⁻stem cells. In certain other embodiments of the method, the placentalstem cells are adherent. In specific embodiments, the adherent placentalstem cells are CD200⁺ and HLA-G⁺; CD73⁺, CD105⁺, and CD200⁺; CD200⁺ andOCT-4⁺; CD73⁺, CD105⁺ and HLA-G⁺; CD73⁺ and CD105⁺ and facilitates theformation of one or more embryoid-like bodies in a population ofplacental cells comprising said stem cell when said population iscultured under conditions that allow the formation of an embryoid-likebody; or OCT-4⁺ and facilitates the formation of one or moreembryoid-like bodies in a population of placental cells comprising thestem cell when said population is cultured under conditions that allowformation of embryoid-like bodies; or any combination thereof. In morespecific embodiments of the nonadherent placental stem cells, theisolated CD200⁺, HLA-G⁺ stem cell is CD34⁻, CD38⁻, CD45⁻, CD73⁺ andCD105⁺; the isolated CD73⁺, CD105⁺, and CD200⁺ stem cell is CD34⁻,CD38⁻, CD45⁻, and HLA-G⁺; the isolated CD200⁺, OCT-4⁺ stem cell isCD34⁻, CD38⁻, CD45⁻, CD73⁺, CD105⁺ and HLA-G⁺; the isolated stem cell ofclaim 1, wherein said CD73⁺, CD105⁺ and HLA-G⁺ stem cell is CD34⁻,CD45⁻, OCT-4⁺ and CD200⁺; the isolated CD73⁺ and CD105⁺ stem cell thatfacilitates the formation of one or more embryoid-like bodies is OCT4⁺,CD34⁻, CD38⁻ and CD45⁻; and/or the isolated OCT-4⁺ and which facilitatesthe formation of one or more embryoid-like bodies is CD73⁺, CD105⁺,CD200⁺, CD34⁻, CD38⁻, and CD45⁻. In certain embodiments, the populationof placental stem cells has been expanded. In certain embodiments, thesaid composition comprises injectable hyaluronic acid. In certainembodiments, the composition comprises injectable collagen. Providedherein are also compositions comprising a population of nonadherent stemcells and injectable hyaluronic acid or collagen.

Placental stem cells can be administered without being cultured underconditions that cause the stem cells to differentiate. Alternately, thestem cells can be cultured in, e.g., e.g., osteogenic medium for, e.g.,about 1-20 days, prior to administration. Alternately, placental stemcells can be isolated and seeded on a matrix, then cultured inosteogenic medium for, e.g., about 1-20 days. In another embodiment,placental stem cells can be cultured in, e.g., osteogenic medium for,e.g., about 1-20 days, then seeded onto a matrix, then cultured inosteogenic medium as described herein for, e.g., about 1-20 days.

In other embodiments, isolated populations of placental stem cells maybe used in autologous or heterologous tissue regeneration or replacementtherapies or protocols, including, but not limited to treatment ofcorneal epithelial defects, cartilage repair, facial dermabrasion,mucosal membranes, tympanic membranes, intestinal linings, neurologicalstructures (e.g., retina, auditory neurons in basilar membrane,olfactory neurons in olfactory epithelium), burn and wound repair fortraumatic injuries of the skin, or for reconstruction of other damagedor diseased organs or tissues.

In certain embodiments, an isolated population of placental stem cellsis used in hematopoietic reconstitution in an individual that hassuffered a partial or total loss of hematopoietic stem cells, e.g.,individuals exposed to lethal or sub-lethal doses of radiation (whetherindustrial, medical or military); individuals that have undergonemyeloablation as part of, e.g., cancer therapy, and the like. Isolatedpopulations of placental-derived stem cells can be used in place of, orto supplement, bone marrow or populations of stem cells derived frombone marrow. Typically, approximately 1×10⁸ to 2×10⁸ bone marrowmononuclear cells per kilogram of patient weight are infused forengraftment in a bone marrow transplantation (i.e., about 70 ml ofmarrow for a 70 kg donor). To obtain 70 ml requires an intensivedonation and significant loss of donor blood in the donation process. Anisolated population of placental stem cells for hematopoieticreconstitution can comprise, in various embodiments, about, at least, orno more than 1×10⁵, 5×10⁵, 1×10⁶, 5×10⁶, 1×10⁷, 5×10⁷, 1×10⁸, 5×10⁸,1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰, 1×10¹¹ or more placental stem cells.

The placental stem cells provided herein, alone or in combination withother stem cell or progenitor cell populations, can be used in themanufacture of a tissue or organ in vivo. The methods provided hereinencompass using cells obtained from the placenta, e.g., stem cells orprogenitor cells, to seed a matrix and to be cultured under theappropriate conditions to allow the cells to differentiate and populatethe matrix. The tissues and organs obtained by the methods providedherein can be used for a variety of purposes, including research andtherapeutic purposes.

In a preferred embodiment, adherent placental stem cells as providedherein, and populations of such stem cells, may be used for autologousand allogenic transplants, including matched and mismatched HLA typehematopoietic transplants. In one embodiment of the use of placentalstem cells as allogenic hematopoietic transplants, the host is treatedto reduce immunological rejection of the donor cells, or to createimmunotolerance (see, e.g., U.S. Pat. Nos. 5,800,539 and 5,806,529). Inanother embodiment, the host is not treated to reduce immunologicalrejection or to create immunotolerance.

Placental stem cells, either alone or in combination with one or moreother stem cell populations, can be used in therapeutic transplantationprotocols, e.g., to augment or replace stem or progenitor cells of theliver, pancreas, kidney, lung, nervous system, muscular system, bone,bone marrow, thymus, spleen, mucosal tissue, gonads, or hair.Additionally, placental stem cells may be used instead of specificclasses of progenitor cells (e.g., chondrocytes, hepatocytes,hematopoietic cells, pancreatic parenchymal cells, neuroblasts, muscleprogenitor cells, etc.) in therapeutic or research protocols in whichprogenitor cells would typically be used.

Placental stem cells as provided herein, and populations of the same,can be used for augmentation, repair or replacement of cartilage,tendon, or ligaments. For example, in certain embodiments, prostheses(e.g., hip prostheses) can be coated with replacement cartilage tissueconstructs grown from placental stem cells provided herein. In otherembodiments, joints (e.g., knee) can be reconstructed with cartilagetissue constructs grown from placental stem cells. Cartilage tissueconstructs can also be employed in major reconstructive surgery fordifferent types of joints (see, e.g., Resnick & Niwayama, eds., 1988,Diagnosis of Bone and Joint Disorders, 2d ed., W. B. Saunders Co.).

The adherent placental stem cells provided herein can be used to repairdamage to tissues and organs resulting from, e.g., trauma, metabolicdisorders, or disease. In such an embodiment, a patient can beadministered placental stem cells, alone or combined with other stem orprogenitor cell populations, to regenerate or restore tissues or organswhich have been damaged as a consequence of disease.

5.8.2 Compositions Comprising Placental Stem Cells

Provided herein are compositions comprising placental stem cells, orbiomolecules therefrom. The adherent placental stem cells providedherein can be combined with any physiologically-acceptable ormedically-acceptable compound, composition or device for use in, e.g.,research or therapeutics.

5.8.2.1 Cryopreserved Placental Stem Cells

The placental stem cell populations provided herein can be preserved,for example, cryopreserved for later use. Methods for cryopreservationof cells, such as stem cells, are well known in the art. Placental stemcell populations can be prepared in a form that is easily administrableto an individual. For example, provided herein is a placental stem cellpopulation that is contained within a container that is suitable formedical use. Such a container can be, for example, a sterile plasticbag, flask, jar, or other container from which the placental stem cellpopulation can be easily dispensed. For example, the container can be ablood bag or other plastic, medically-acceptable bag suitable for theintravenous administration of a liquid to a recipient. The container ispreferably one that allows for cryopreservation of the combined stemcell population.

The cryopreserved placental stem cell population can comprise placentalstem cells derived from a single donor, or from multiple donors. Theplacental stem cell population can be completely HLA-matched to anintended recipient, or partially or completely HLA-mismatched.

Thus, in one embodiment, provided herein is a composition comprising aplacental stem cell population in a container. In a specific embodiment,the stem cell population is cryopreserved. In another specificembodiment, the container is a bag, flask, or jar. In more specificembodiment, said bag is a sterile plastic bag. In a more specificembodiment, said bag is suitable for, allows or facilitates intravenousadministration of said placental stem cell population. The bag cancomprise multiple lumens or compartments that are interconnected toallow mixing of the placental stem cells and one or more othersolutions, e.g., a drug, prior to, or during, administration. In anotherspecific embodiment, the composition comprises one or more compoundsthat facilitate cryopreservation of the combined stem cell population.In another specific embodiment, said placental stem cell population iscontained within a physiologically-acceptable aqueous solution. In amore specific embodiment, said physiologically-acceptable aqueoussolution is a 0.9% NaCl solution. In another specific embodiment, saidplacental stem cell population comprises placental cells that areHLA-matched to a recipient of said stem cell population. In anotherspecific embodiment, said combined stem cell population comprisesplacental cells that are at least partially HLA-mismatched to arecipient of said stem cell population. In another specific embodiment,said placental stem cells are derived from a plurality of donors.

5.8.2.2 Pharmaceutical Compositions

Populations of placental stem cells, or populations of cells comprisingplacental stem cells, can be formulated into pharmaceutical compositionsfor use in vivo. Such pharmaceutical compositions comprise a populationof placental stem cells, or a population of cells comprising placentalstem cells, in a pharmaceutically-acceptable carrier, e.g., a salinesolution or other accepted physiologically-acceptable solution for invivo administration. Pharmaceutical compositions provided herein cancomprise any of the placental stem cell populations, or placental stemcell types, described elsewhere herein. The pharmaceutical compositionscan comprise fetal, maternal, or both fetal and maternal placental stemcells. The pharmaceutical compositions provided herein can furthercomprise placental stem cells obtained from a single individual orplacenta, or from a plurality of individuals or placentae.

The pharmaceutical compositions provided herein can comprise any numberof placental stem cells. For example, a single unit dose of placentalstem cells can comprise, in various embodiments, about, at least, or nomore than 1×10⁵, 5×10⁵, 1×10⁶, 5×10⁶, 1×10⁷, 5×10⁷, 1×10⁸, 5×10⁸, 1×10⁹,5×10⁹, 1×10¹⁰, 5×10¹⁰, 1×10¹¹ or more placental stem cells.

The pharmaceutical compositions provided herein can comprise populationsof cells that comprise 50% viable cells or more (that is, at least about50% of the cells in the population are functional or living).Preferably, at least about 60% of the cells in the population areviable. More preferably, at least about 70%, 80%, 90%, 95%, or 99% ofthe cells in the population in the pharmaceutical composition areviable.

The pharmaceutical compositions provided herein can comprise one or morecompounds that, e.g., facilitate engraftment (e.g., anti-T-cell receptorantibodies, an immunosuppressant, or the like); stabilizers such asalbumin, dextran 40, gelatin, hydroxyethyl starch, and the like.

5.8.2.3 Placental Stem Cell Conditioned Media

The placental stem cells provided herein can be used to produceconditioned medium, that is, medium comprising one or more biomoleculessecreted or excreted by the stem cells. In various embodiments, theconditioned medium comprises medium in which placental stem cells havegrown for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or moredays. In other embodiments, the conditioned medium comprises medium inwhich placental stem cells have grown to at least about 30%, 40%, 50%,60%, 70%, 80%, 90% confluence, or up to 100% confluence. Suchconditioned medium can be used to support the culture of a separatepopulation of placental stem cells, or stem cells of another kind. Inanother embodiment, the conditioned medium comprises medium in whichplacental stem cells have been differentiated into an adult cell type.In another embodiment, the conditioned medium provided herein comprisesmedium in which placental stem cells and non-placental stem cells havebeen cultured.

5.8.2.4 Matrices Comprising Placental Stem Cells

Further provided herein are matrices, hydrogels, scaffolds, and the likethat comprise a placental stem cell, or a population of placental stemcells. In certain embodiments, the matrix can be any substrate known toone skilled in the art to be useful for treating bone defects. Forexample, the matrix can be a β-tricalcium phosphate substrate, aβ-tricalcium phosphate-collagen substrate, a collagen substrate, acalcium phosphate substrate, a mineralized collagen substrate, and ahyaluronic acid substrate. In some embodiments, the collagen in thematrix can be placental collagen. Methods and compositions for isolatingand preparing placental collagen are extensively described, for example,in U.S. patent application Ser. No. 11/450,934, filed Jun. 9, 2006.

Placental stem cells can be seeded onto the matrix for treating boneprior to or after a differentiation step. For example, placental stemcells can be cultured in, e.g., osteogenic medium for, e.g., about 1-20days, then seeded onto the matrix. Alternately, placental stem cells canbe isolated and seeded onto the matrix, then cultured in osteogenicmedium as described herein for, e.g., about 1-20 days. In anotherembodiment, placental stem cells are cultured in, e.g., osteogenicmedium for, e.g., about 1-20 days, then seeded onto the matrix, thencultured in osteogenic medium as described herein for, e.g., about 1-20days.

Placental stem cells can be seeded onto a natural matrix, e.g., aplacental biomaterial such as an amniotic membrane material. Such anamniotic membrane material can be, e.g., amniotic membrane dissecteddirectly from a mammalian placenta; fixed or heat-treated amnioticmembrane, substantially dry (i.e., <20% H₂O) amniotic membrane,chorionic membrane, substantially dry chorionic membrane, substantiallydry amniotic and chorionic membrane, and the like. Preferred placentalbiomaterials on which placental stem cells can be seeded are describedin Hariri, U.S. Application Publication No. 2004/0048796.

Placental stem cells as provided herein can be suspended in a hydrogelsolution suitable for, e.g., injection. Suitable hydrogels for suchcompositions include self-assembling peptides, such as RAD16. In oneembodiment, a hydrogel solution comprising the cells can be allowed toharden, for instance in a mold, to form a matrix having cells dispersedtherein for implantation. Placental stem cells in such a matrix can alsobe cultured so that the cells are mitotically expanded prior toimplantation. The hydrogel is, e.g., an organic polymer (natural orsynthetic) that is cross-linked via covalent, ionic, or hydrogen bondsto create a three-dimensional open-lattice structure that entraps watermolecules to form a gel. Hydrogel-forming materials includepolysaccharides such as alginate and salts thereof, peptides,polyphosphazines, and polyacrylates, which are crosslinked ionically, orblock polymers such as polyethylene oxide-polypropylene glycol blockcopolymers which are crosslinked by temperature or pH, respectively. Insome embodiments, the hydrogel or matrix biodegradable.

In some embodiments, the formulation comprises an in situ polymerizablegel (see., e.g., U.S. Patent Application Publication 2002/0022676;Anseth et al., J. Control Release, 78(1-3):199-209 (2002); Wang et al.,Biomaterials, 24(22):3969-80 (2003).

In some embodiments, the polymers are at least partially soluble inaqueous solutions, such as water, buffered salt solutions, or aqueousalcohol solutions, that have charged side groups, or a monovalent ionicsalt thereof. Examples of polymers having acidic side groups that can bereacted with cations are poly(phosphazenes), poly(acrylic acids),poly(methacrylic acids), copolymers of acrylic acid and methacrylicacid, poly(vinyl acetate), and sulfonated polymers, such as sulfonatedpolystyrene. Copolymers having acidic side groups formed by reaction ofacrylic or methacrylic acid and vinyl ether monomers or polymers canalso be used. Examples of acidic groups are carboxylic acid groups,sulfonic acid groups, halogenated (preferably fluorinated) alcoholgroups, phenolic OH groups, and acidic OH groups.

The placental stem cells or co-cultures thereof can be seeded onto athree-dimensional framework or scaffold and implanted in vivo. Such aframework can be implanted in combination with any one or more growthfactors, cells, drugs or other components that stimulate tissueformation or otherwise enhance or improve repair of tissue.

Examples of scaffolds that can be used include nonwoven mats, porousfoams, or self assembling peptides. Nonwoven mats can be formed usingfibers comprised of a synthetic absorbable copolymer of glycolic andlactic acids (e.g., PGA/PLA) (VICRYL, Ethicon, Inc., Somerville, N.J.).Foams, composed of, e.g., poly(ε-caprolactone)/poly(glycolic acid)(PCL/PGA) copolymer, formed by processes such as freeze-drying, orlyophilization (see, e.g., U.S. Pat. No. 6,355,699), can also be used asscaffolds.

Placental stem cells provided herein can also be seeded onto, orcontacted with, a physiologically-acceptable ceramic material including,but not limited to, mono-, di-, tri-, alpha-tri-, beta-tri-, andtetra-calcium phosphate, hydroxyapatite, fluoroapatites, calciumsulfates, calcium fluorides, calcium oxides, calcium carbonates,magnesium calcium phosphates, biologically active glasses such asBIOGLASS®, and mixtures thereof. Porous biocompatible ceramic materialscurrently commercially available include SURGIBONE® (CanMedica Corp.,Canada), ENDOBON® (Merck Biomaterial France, France), CEROS® (Mathys,AG, Bettlach, Switzerland), and mineralized collagen bone graftingproducts such as HEALOS™ (DePuy, Inc., Raynham, Mass.) and VITOSS®,RHAKOSS™, and CORTOSS® (Orthovita, Malvern, Pa.). The framework can be amixture, blend or composite of natural and/or synthetic materials.

In another embodiment, placental stem cells can be seeded onto, orcontacted with, a felt, which can be, e.g., composed of a multifilamentyarn made from a bioabsorbable material such as PGA, PLA, PCL copolymersor blends, or hyaluronic acid.

The placental stem cells provided herein can, in another embodiment, beseeded onto foam scaffolds that may be composite structures. Such foamscaffolds can be molded into a useful shape, such as that of a portionof a specific structure in the body to be repaired, replaced oraugmented. In some embodiments, the framework is treated, e.g., with0.1M acetic acid followed by incubation in polylysine, PBS, and/orcollagen, prior to inoculation of the placental stem cells in order toenhance cell attachment. External surfaces of a matrix may be modifiedto improve the attachment or growth of cells and differentiation oftissue, such as by plasma-coating the matrix, or addition of one or moreproteins (e.g., collagens, elastic fibers, reticular fibers),glycoproteins, glycosaminoglycans (e.g., heparin sulfate,chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratinsulfate, etc.), a cellular matrix, and/or other materials such as, butnot limited to, gelatin, alginates, agar, agarose, and plant gums, andthe like.

In some embodiments, the scaffold comprises, or is treated with,materials that render it non-thrombogenic. These treatments andmaterials may also promote and sustain endothelial growth, migration,and extracellular matrix deposition. Examples of these materials andtreatments include but are not limited to natural materials such asbasement membrane proteins such as laminin and Type IV collagen,synthetic materials such as EPTFE, and segmented polyurethaneureasilicones, such as PURSPAN™ (The Polymer Technology Group, Inc.,Berkeley, Calif.). The scaffold can also comprise anti-thrombotic agentssuch as heparin; the scaffolds can also be treated to alter the surfacecharge (e.g., coating with plasma) prior to seeding with placental stemcells. The scaffold can further comprise agents that stimulate bonegrowth and/or inhibit bone resorption. For example, the scaffold cancomprise bone morphogenic proteins, e.g., BMP-2 and/or BMP-7, WNTinhibitors, and the like.

5.8.3 Immortalized Placental Stem Cell Lines

Mammalian placental cells can be conditionally immortalized bytransfection with any suitable vector containing a growth-promotinggene, that is, a gene encoding a protein that, under appropriateconditions, promotes growth of the transfected cell, such that theproduction and/or activity of the growth-promoting protein isregulatable by an external factor. In a preferred embodiment thegrowth-promoting gene is an oncogene such as, but not limited to, v-myc,N-myc, c-myc, p53, SV40 large T antigen, polyoma large T antigen, Elaadenovirus or E7 protein of human papillomavirus.

External regulation of the growth-promoting protein can be achieved byplacing the growth-promoting gene under the control of anexternally-regulatable promoter, e.g., a promoter the activity of whichcan be controlled by, for example, modifying the temperature of thetransfected cells or the composition of the medium in contact with thecells. in one embodiment, a tetracycline (tet)-controlled geneexpression system can be employed (see Gossen et al., Proc. Natl. Acad.Sci. USA 89:5547-5551, 1992; Hoshimaru et al., Proc. Natl. Acad. Sci.USA 93:1518-1523, 1996). In the absence of tet, a tet-controlledtransactivator (tTA) within this vector strongly activates transcriptionfrom ph_(CMV) ₊ ⁻¹, a minimal promoter from human cytomegalovirus fusedto tet operator sequences. tTA is a fusion protein of the repressor(tetR) of the transposon-10-derived tet resistance operon of Escherichiacoli and the acidic domain of VP16 of herpes simplex virus. Low,non-toxic concentrations of tet (e.g., 0.01-1.0 μg/mL) almost completelyabolish transactivation by tTA.

In one embodiment, the vector further contains a gene encoding aselectable marker, e.g., a protein that confers drug resistance. Thebacterial neomycin resistance gene (neo^(R)) is one such marker that maybe employed as described herein. Cells carrying neo^(R) may be selectedby means known to those of ordinary skill in the art, such as theaddition of, e.g., 100-200 μg/mL G418 to the growth medium.

Transfection can be achieved by any of a variety of means known to thoseof ordinary skill in the art including, but not limited to, retroviralinfection. In general, a cell culture may be transfected by incubationwith a mixture of conditioned medium collected from the producer cellline for the vector and DMEM/F12 containing N2 supplements. For example,a placental cell culture prepared as described above may be infectedafter, e.g., five days in vitro by incubation for about 20 hours in onevolume of conditioned medium and two volumes of DMEM/F12 containing N2supplements. Transfected cells carrying a selectable marker may then beselected as described above.

Following transfection, cultures are passaged onto a surface thatpermits proliferation, e.g., allows at least about 30% of the cells todouble in a 24 hour period. Preferably, the substrate is apolyornithine/laminin substrate, consisting of tissue culture plasticcoated with polyornithine (10 μg/mL) and/or laminin (10 μg/mL), apolylysine/laminin substrate or a surface treated with fibronectin.Cultures are then fed every 3-4 days with growth medium, which may ormay not be supplemented with one or more proliferation-enhancingfactors. Proliferation-enhancing factors may be added to the growthmedium when cultures are less than 50% confluent.

The conditionally-immortalized placental stem cell lines can be passagedusing standard techniques, such as by trypsinization, when 80-95%confluent. Up to approximately the twentieth passage, it is, in someembodiments, beneficial to maintain selection (by, for example, theaddition of G418 for cells containing a neomycin resistance gene). Cellsmay also be frozen in liquid nitrogen for long-term storage.

Clonal cell lines can be isolated from a conditionally-immortalizedhuman placental stem cell line prepared as described above. In general,such clonal cell lines may be isolated using standard techniques, suchas by limit dilution or using cloning rings, and expanded. Clonal celllines may generally be fed and passaged as described above.

Conditionally-immortalized human placental stem cell lines, which may,but need not, be clonal, may generally be induced to differentiate bysuppressing the production and/or activity of the growth-promotingprotein under culture conditions that facilitate differentiation. Forexample, if the gene encoding the growth-promoting protein is under thecontrol of an externally-regulatable promoter, the conditions, e.g.,temperature or composition of medium, may be modified to suppresstranscription of the growth-promoting gene. For thetetracycline-controlled gene expression system discussed above,differentiation can be achieved by the addition of tetracycline tosuppress transcription of the growth-promoting gene. In general, 1 μg/mLtetracycline for 4-5 days is sufficient to initiate differentiation. Topromote further differentiation, additional agents may be included inthe growth medium.

5.8.4 Assays

The placental stem cells provided herein can be used in assays todetermine the influence of culture conditions, environmental factors,molecules (e.g., biomolecules, small inorganic molecules. etc.) and thelike on stem cell proliferation, expansion, and/or differentiation,compared to placental stem cells not exposed to such conditions.

In a preferred embodiment, the placental stem cells provided herein areassayed for changes in proliferation, expansion or differentiation uponcontact with a molecule. For example, osteogenic differentiation can beassayed by monitoring alkaline phosphatase activity and/or calciummineralization.

In one embodiment, for example, provided herein is a method ofidentifying a compound that modulates the proliferation of a pluralityof placental stem cells, comprising contacting said plurality of stemcells with said compound under conditions that allow proliferation,wherein if said compound causes a detectable change in proliferation ofsaid plurality of stem cells compared to a plurality of stem cells notcontacted with said compound, said compound is identified as a compoundthat modulates proliferation of placental stem cells. In a specificembodiment, said compound is identified as an inhibitor ofproliferation. In another specific embodiment, said compound isidentified as an enhancer of proliferation.

In another embodiment, provided herein is a method of identifying acompound that modulates the expansion of a plurality of placental stemcells, comprising contacting said plurality of stem cells with saidcompound under conditions that allow expansion, wherein if said compoundcauses a detectable change in expansion of said plurality of stem cellscompared to a plurality of stem cells not contacted with said compound,said compound is identified as a compound that modulates expansion ofplacental stem cells. In a specific embodiment, said compound isidentified as an inhibitor of expansion. In another specific embodiment,said compound is identified as an enhancer of expansion.

In another embodiment, provided herein is a method of identifying acompound that modulates the differentiation of a placental stem cell,comprising contacting said stem cells with said compound underconditions that allow differentiation, wherein if said compound causes adetectable change in differentiation of said stem cells compared to astem cell not contacted with said compound, said compound is identifiedas a compound that modulates proliferation of placental stem cells. In aspecific embodiment, said compound is identified as an inhibitor ofdifferentiation. In another specific embodiment, said compound isidentified as an enhancer of differentiation.

6. EXAMPLES

The following examples are intended to illustrate the presentembodiments and are not to be construed to be limiting in any way. Allreferences, whether patent references, literature references, orotherwise, cited herein are hereby incorporated by reference for allpurposes.

6.1 Example 1: Culture of Placental Stem Cells

Placental stem cells are obtained from a post-partum mammalian placentaeither by perfusion or by physical disruption, e.g., enzymaticdigestion. The cells are cultured in a culture medium comprising 60%DMEM-LG (Gibco), 40% MCDB-201(Sigma), 2% fetal calf serum (FCS) (HycloneLaboratories), lx insulin-transferrin-selenium (ITS), lxlenolenic-acid-bovine-serum-albumin (LA-BSA), 10⁻⁹M dexamethasone(Sigma), 10⁻⁴M ascorbic acid 2-phosphate (Sigma), epidermal growthfactor (EGF)10 ng/ml (R&D Systems), platelet derived-growth factor(PDGF-BB) 10 ng/ml (R&D Systems), and 100U penicillin/1000Ustreptomycin.

The culture flask in which the cells are cultured is prepared asfollows. T75 flasks are coated with fibronectin (FN), by adding 5 ml PBScontaining 5 ng/ml human FN (Sigma F0895) to the flask. The flasks withFN solution are left at 37° C. for 30 min. The FN solution is thenremoved prior to cell culture. There is no need to dry the flasksfollowing treatment. Alternatively, the flasks are left in contact withthe FN solution at 4° C. overnight or longer; prior to culture, theflasks are warmed and the FN solution is removed.

Placental Stem Cells Isolated by Perfusion

Cultures of placental stem cells from placental perfusate areestablished as follows. Cells from a Ficoll gradient are seeded inFN-coated T75 flasks, prepared as above, at 50-100×10⁶ cells/flask in 15ml culture medium. Typically, 5 to 10 flasks are seeded. The flasks areincubated at 37° C. for 12-18 hrs to allow the attachment of adherentcells. 10 ml of warm PBS is added to each flask to remove cells insuspension, and mixed gently. 15 mL of the medium is then removed andreplaced with 15 ml fresh culture medium. All medium is changed 3-4 daysafter the start of culture. Subsequent culture medium changes areperformed, during which 50% or 7.5 ml of the medium is removed.

Starting at about day 12, the culture is checked under a microscope toexamine the growth of the adherent cell colonies. When cell culturesbecome approximately 80% confluent, typically between day 13 to day 18after the start of culture, adherent cells are harvested by trypsindigestion. Cells harvested from these primary cultures are designatedpassage 0 (zero).

Placental Stem Cells Isolated by Physical Disruption and EnzymaticDigestion

Placental stem cell cultures are established from digested placentaltissue as follows. The perfused placenta is placed on a sterile papersheet with the maternal side up. Approximately 0.5 cm of the surfacelayer on maternal side of placenta is scraped off with a blade, and theblade is used to remove a placental tissue block measuring approximately1×2×1 cm. This placenta tissue is then minced into approximately 1 mm³pieces. These pieces are collected into a 50 ml Falcon tube and digestedwith collagenase IA (2 mg/ml, Sigma) for 30 minutes, followed bytrypsin-EDTA (0.25%, GIBCO BRL) for 10 minutes, at 37° C. in water bath.The resulting solution is centrifuged at 400 g for 10 minutes at roomtemperature, and the digestion solution is removed. The pellet isresuspended to approximately 10 volumes with PBS (for example, a 5 mlpellet is resuspended with 45 ml PBS), and the tubes are centrifuged at400 g for 10 minutes at room temperature. The tissue/cell pellet isresuspended in 130 mL culture medium, and the cells are seeded at 13 mlper fibronectin-coated T-75 flask. Cells are incubated at 37° C. with ahumidified atmosphere with 5% CO₂. Placental Stem Cells are optionallycryopreserved at this stage.

Subculturing and Expansion of Placental Stem Cells

Cryopreserved cells are quickly thawed in a 37° C. water bath. Placentalstem cells are immediately removed from the cryovial with 10 ml warmmedium and transferred to a 15 ml sterile tube. The cells arecentrifuged at 400 g for 10 minutes at room temperature. The cells aregently resuspended in 10 ml of warm culture medium by pipetting, andviable cell counts are determined by Trypan blue exclusion. Cells arethen seeded at about 6000-7000 cells per cm² onto FN-coated flasks,prepared as above (approximately 5×10⁵ cells per T-75 flask). The cellsare incubated at 37° C., 5% CO₂ and 90% humidity. When the cells reached75-85% confluency, all of the spent media is aseptically removed fromthe flasks and discarded. 3 ml of 0.25% trypsin/EDTA (w/v) solution isadded to cover the cell layer, and the cells are incubated at 37° C., 5%CO₂ and 90% humidity for 5 minutes. The flask is tapped once or twice toexpedite cell detachment. Once >95% of the cells are rounded anddetached, 7 ml of warm culture medium is added to each T-75 flask, andthe solution is dispersed by pipetting over the cell layer surfaceseveral times.

After counting the cells and determining viability as above, the cellsare centrifuged at 1000 RPM for 5 minutes at room temperature. Cells arepassaged by gently resuspending the cell pellet from one T-75 flask withculture medium, and evenly plating the cells onto two FN-coated T-75flasks.

Using the above methods, populations of adherent placental stem cellsare identified that express markers CD105, CD117, CD33, CD73, CD29,CD44, CD10, CD90 and CD133. This population of cells did not expressCD34 or CD45. Some, but not all cultures of these placental stem cellsexpressed HLA-ABC and/or HLA-DR.

6.2 Example 2: Isolation of Placental Stem Cells from PlacentalStructures 6.2.1 Materials & Methods 6.2.1.1 Isolation of the Phenotypeof Interest

Five distinct populations of placental cells were obtained from theplacentas of normal, full-term pregnancies. All donors provided fullwritten consent for the use of their placentas for research purposes.Five populations of placental cells were examined: (1) placentalperfusate (from perfusion of the placental vasculature); and enzymaticdigestions of (2) amnion, (3) chorion, (4) amnion-chorion plate, and (5)umbilical cord. The various placental tissues were cleaned in sterilePBS (Gibco-Invitrogen Corporation, Carlsbad, Calif.) and placed onseparate sterile Petri dishes. The various tissues were minced using asterile surgical scalpel and placed into 50 mL Falcon Conical tubes. Theminced tissues were digested with 1× Collagenase (Sigma-Aldrich, St.Louis, Mo.) for 20 minutes in a 37° C. water bath, centrifuged, and thendigested with 0.25% Trypsin-EDTA (Gibco-Invitrogen Corp) for 10 minutesin a 37° C. water bath. The various tissues were centrifuged afterdigestion and rinsed once with sterile PBS (Gibco-Invitrogen Corp). Thereconstituted cells were then filtered twice, once with 100 μm cellstrainers and once with 30 μm separation filters, to remove any residualextracellular matrix or cellular debris.

6.2.1.2 Cellular Viability Assessment and Cell Counts

The manual trypan blue exclusion method was employed post digestion tocalculate cell counts and assess cellular viability. Cells were mixedwith Trypan Blue Dye (Sigma-Aldrich) at a ratio of 1:1, and the cellswere read on hemacytometer.

6.2.1.3 Cell Surface Marker Characterization

Cells that were HLA ABC⁻/CD45⁻/CD34⁻/CD133⁺ were selected forcharacterization. Cells having this phenotype were identified,quantified, and characterized by two of Becton-Dickinson flowcytometers, the FACSCalibur and the FACS Aria (Becton-Dickinson, SanJose, Calif., USA). The various placental cells were stained, at a ratioof about 10 μL of antibody per 1 million cells, for 30 minutes at roomtemperature on a shaker. The following anti-human antibodies were used:Fluorescein Isothiocyanate (FITC) conjugated monoclonal antibodiesagainst HLA-G (Serotec, Raleigh, N.C.), CD10 (BD ImmunocytometrySystems, San Jose, Calif.), CD44 (BD Biosciences Pharmingen, San Jose,Calif.), and CD105 (R&D Systems Inc., Minneapolis, Minn.); Phycoerythrin(PE) conjugated monoclonal antibodies against CD44, CD200, CD117, andCD13 (BD Biosciences Pharmingen); Phycoerythrin-Cy5 (PE Cy5) conjugatedStreptavidin and monoclonal antibodies against CD117 (BD BiosciencesPharmingen); Phycoerythrin-Cy7 (PE Cy7) conjugated monoclonal antibodiesagainst CD33 and CD10 (BD Biosciences); Allophycocyanin (APC) conjugatedstreptavidin and monoclonal antibodies against CD38 (BD BiosciencesPharmingen); and Biotinylated CD90 (BD Biosciences Pharmingen). Afterincubation, the cells were rinsed once to remove unbound antibodies andwere fixed overnight with 4% paraformaldehyde (USB, Cleveland, Ohio) at4° C. The following day, the cells were rinsed twice, filtered through a30 μm separation filter, and were run on the flow cytometer(s).

Samples that were stained with anti-mouse IgG antibodies (BD BiosciencesPharmingen) were used as negative controls and were used to adjust thePhoto Multiplier Tubes (PMTs). Samples that were single stained withanti-human antibodies were used as positive controls and were used toadjust spectral overlaps/compensations.

6.2.1.4 Cell Sorting and Culture

One set of placental cells (from perfusate, amnion, or chorion) wasstained with 7-Amino-Actinomycin D (7AAD; BD Biosciences Pharmingen) andmonoclonal antibodies specific for the phenotype of interest. The cellswere stained at a ratio of 10 μL of antibody per 1 million cells, andwere incubated for 30 minutes at room temperature on a shaker. Thesecells were then positively sorted for live cells expressing thephenotype of interest on the BD FACS Aria and plated into culture.Sorted (population of interest) and “All” (non-sorted) placental cellpopulations were plated for comparisons. The cells were plated onto afibronectin (Sigma-Aldrich) coated 96 well plate at the cell densitieslisted in Table 1 (cells/cm²). The cell density, and whether the celltype was plated in duplicate or triplicate, was determined and governedby the number of cells expressing the phenotype of interest.

TABLE 1 Cell plating densities 96 Well Plate Culture Density of PlatedCells Conditions Sorted All All Max. Density Cell Source A Set #1: 40.6K/cm² 40.6 K/cm² 93.8 K/cm² Set #2 40.6 K/cm² 40.6 K/cm² 93.8 K/cm² Set#3: 40.6 K/cm² 40.6 K/cm² 93.8 K/cm² Cell Source B Set #1: 6.3 K/cm² 6.3K/cm² 62.5 K/cm² Set #2 6.3 K/cm² 6.3 K/cm² 62.5 K/cm² Cell Source C Set#1: 6.3 K/cm² 6.3 K/cm² 62.5 K/cm² Set #2 6.3 K/cm² 6.3 K/cm² 62.5 K/cm²

Complete medium (60% DMEM-LG (Gibco) and 40% MCDB-201 (Sigma); 2% fetalcalf serum (Hyclone Labs.); lx insulin-transferrin-selenium (ITS); lxlinoleic acid-bovine serum albumin (LA-BSA); 10⁻⁹ M dexamethasone(Sigma); 10⁴ M ascorbic acid 2-phosphate (Sigma); epidermal growthfactor 10 ng/mL (R&D Systems); and platelet-derived growth factor(PDGF-BB) 10 ng/mL (R&D Systems)) was added to each well of the 96 wellplate and the plate was placed in a 5% CO₂/37° C. incubator. On day 7,100 μL of complete medium was added to each of the wells. The 96 wellplate was monitored for about two weeks and a final assessment of theculture was completed on day 12.

6.2.1.5 Data Analysis

FACSCalibur data was analyzed in FlowJo (Tree star, Inc) using standardgating techniques. The BD FACS Aria data was analyzed using the FACSDivasoftware (Becton-Dickinson). The FACS Aria data was analyzed usingdoublet discrimination gating to minimize doublets, as well as, standardgating techniques. All results were compiled in Microsoft Excel and allvalues, herein, are represented as average±standard deviation (number,standard error of mean).

6.2.2 Results 6.2.2.1 Cellular Viability

Post-digestion viability was assessed using the manual trypan blueexclusion method (FIG. 1). The average viability of cells obtained fromthe majority of the digested tissue (from amnion, chorion oramnion-chorion plate) was around 70%. Amnion had an average viability of74.35%±10.31% (n=6, SEM=4.21), chorion had an average viability of78.18%±12.65% (n=4, SEM=6.32), amnion-chorion plate had an averageviability of 69.05%±10.80% (n=4, SEM=5.40), and umbilical cord had anaverage viability of 63.30%±20.13% (n=4, SEM=10.06). Cells fromperfusion, which did not undergo digestion, retained the highest averageviability, 89.98±6.39% (n=5, SEM=2.86).

6.2.2.2 Cell Quantification

The five distinct populations of placenta derived cells were analyzed todetermine the numbers of HLA ABC⁻/CD45⁻/CD34⁻/CD133⁺ cells. From theanalysis of the BD FACSCalibur data, it was observed that the amnion,perfusate, and chorion contained the greatest total number of thesecells, 30.72±21.80 cells (n=4, SEM=10.90), 26.92±22.56 cells (n=3,SEM=13.02), and 18.39±6.44 cells (n=2, SEM=4.55) respectively (data notshown). The amnion-chorion plate and umbilical cord contained the leasttotal number of cells expressing the phenotype of interest, 4.72±4.16cells (n=3, SEM=2.40) and 3.94±2.58 cells (n=3, SEM=1.49) respectively(data not shown).

Similarly, when the percent of total cells expressing the phenotype ofinterest was analyzed, it was observed that amnion and placentalperfusate contained the highest percentages of cells expressing thisphenotype (0.0319%±0.0202% (n=4, SEM=0.0101) and 0.0269%±0.0226% (n=3,SEM=0.0130) respectively (FIG. 2). Although umbilical cord contained asmall number of cells expressing the phenotype of interest (FIG. 2), itcontained the third highest percentage of cells expressing the phenotypeof interest, 0.020±0.0226% (n=3, SEM=0.0131) (FIG. 2). The chorion andamnion-chorion plate contained the lowest percentages of cellsexpressing the phenotype of interest, 0.0184±0.0064% (n=2, SEM=0.0046)and 0.0177±0.0173% (n=3, SEM=0.010) respectively (FIG. 2).

Consistent with the results of the BD FACSCalibur analysis, the BD FACSAria data also identified amnion, perfusate, and chorion as providinghigher numbers of HLA ABC⁻/CD45⁻/CD34⁻/CD133⁺ cells than the remainingsources. The average total number of cells expressing the phenotype ofinterest among amnion, perfusate, and chorion was 126.47±55.61 cells(n=15, SEM=14.36), 81.65±34.64 cells (n=20, SEM=7.75), and 51.47±32.41cells (n=15, SEM=8.37), respectively (data not shown). Theamnion-chorion plate and umbilical cord contained the least total numberof cells expressing the phenotype of interest, 44.89±37.43 cells (n=9,SEM=12.48) and 11.00±4.03 cells (n=9, SEM=1.34) respectively (data notshown).

BD FACS Aria data revealed that the B and A cell sources contained thehighest percentages of HLA ABC⁻/CD45⁻/CD34⁻/CD133⁺ cells, 0.1523±0.0227%(n=15, SEM=0.0059) and 0.0929±0.0419% (n=20, SEM=0.0094) respectively(FIG. 3). The D cell source contained the third highest percentage ofcells expressing the phenotype of interest, 0.0632±0.0333% (n=9,SEM=0.0111) (FIG. 3). The C and E cell sources contained the lowestpercentages of cells expressing the phenotype of interest,0.0623±0.0249% (n=15, SEM=0.0064) and 0.0457±0.0055% (n=9, SEM=0.0018)respectively (FIG. 3).

After HLA ABC⁻/CD45⁻/CD34⁻/CD133⁺ cells were identified and quantifiedfrom each cell source, its cells were further analyzed and characterizedfor their expression of cell surface markers HLA-G, CD10, CD13, CD33,CD38, CD44, CD90, CD105, CD117, CD200, and CD105.

6.2.2.3 Placental Perfusate Derived Cells

Perfusate-derived cells were consistently positive for HLA-G, CD33,CD117, CD10, CD44, CD200, CD90, CD38, CD105, and CD13 (FIG. 4). Theaverage expression of each marker for perfusate-derived cells was thefollowing: 37.15%±38.55% (n=4, SEM=19.28) of the cells expressed HLA-G;36.37%±21.98% (n=7, SEM=8.31) of the cells expressed CD33; 39.39%±39.91%(n=4, SEM=19.96) of the cells expressed CD117; 54.97%±33.08% (n=4,SEM=16.54) of the cells expressed CD10; 36.79%±11.42% (n=4, SEM=5.71) ofthe cells expressed CD44; 41.83%±19.42% (n=3, SEM=11.21) of the cellsexpressed CD200; 74.25%±26.74% (n=3, SEM=15.44) of the cells expressedCD90; 35.10%±23.10% (n=3, SEM=13.34) of the cells expressed CD38;22.87%±6.87% (n=3, SEM=3.97) of the cells expressed CD105; and25.49%±9.84% (n=3, SEM=5.68) of the cells expressed CD13.

6.2.2.4 Amnion-Derived Cells

Amnion-derived cells were consistently positive for HLA-G, CD33, CD117,CD10, CD44, CD200, CD90, CD38, CD105, and CD13 (FIG. 5). The averageexpression of each marker for amnion-derived was the following:57.27%±41.11% (n=3, SEM=23.73) of the cells expressed HLA-G;16.23%±15.81% (n=6, SEM=6.46) of the cells expressed CD33; 62.32%±37.89%(n=3, SEM=21.87) of the cells expressed CD117; 9.71%±13.73% (n=3,SEM=7.92) of the cells expressed CD10; 27.03%±22.65% (n=3, SEM=13.08) ofthe cells expressed CD44; 6.42%±0.88% (n=2, SEM=0.62) of the cellsexpressed CD200; 57.61%±22.10% (n=2, SEM=15.63) of the cells expressedCD90; 63.76%±4.40% (n=2, SEM=3.11) of the cells expressed CD38;20.27%±5.88% (n=2, SEM=4.16) of the cells expressed CD105; and54.37%±13.29% (n=2, SEM=9.40) of the cells expressed CD13.

6.2.2.5 Chorion-Derived Cells

Chorion-derived cells were consistently positive for HLA-G, CD117, CD10,CD44, CD200, CD90, CD38, and CD13, while the expression of CD33, andCD105 varied (FIG. 6). The average expression of each marker for chorioncells was the following: 53.25%±32.87% (n=3, SEM=18.98) of the cellsexpressed HLA-G; 15.44%±11.17% (n=6, SEM=4.56) of the cells expressedCD33; 70.76%±11.87% (n=3, SEM=6.86) of the cells expressed CD117;35.84%±25.96% (n=3, SEM=14.99) of the cells expressed CD10; 28.76%±6.09%(n=3, SEM=3.52) of the cells expressed CD44; 29.20%±9.47% (n=2,SEM=6.70) of the cells expressed CD200; 54.88%±0.17% (n=2, SEM=0.12) ofthe cells expressed CD90; 68.63%±44.37% (n=2, SEM=31.37) of the cellsexpressed CD38; 23.81%±33.67% (n=2, SEM=23.81) of the cells expressedCD105; and 53.16%±62.70% (n=2, SEM=44.34) of the cells expressed CD13.

6.2.2.6 Source D Placental Cells

Cells from amnion-chorion plate were consistently positive for HLA-G,CD33, CD117, CD10, CD44, CD200, CD90, CD38, CD105, and CD13 (FIG. 7).The average expression of each marker for amnion-chorion plate-derivedcells was the following: 78.52%±13.13% (n=2, SEM=9.29) of the cellsexpressed HLA-G; 38.33%±15.74% (n=5, SEM=7.04) of the cells expressedCD33; 69.56%±26.41% (n=2, SEM=18.67) of the cells expressed CD117;42.44%±53.12% (n=2, SEM=37.56) of the cells expressed CD10;32.47%±31.78% (n=2, SEM=22.47) of the cells expressed CD44; 5.56% (n=1)of the cells expressed CD200; 83.33% (n=1) of the cells expressed CD90;83.52% (n=1) of the cells expressed CD38; 7.25% (n=1) of the cellsexpressed CD105; and 81.16% (n=1) of the cells expressed CD13.

6.2.2.7 Umbilical Cord-Derived Cells

Umbilical cord-derived cells were consistently positive for HLA-G, CD33,CD90, CD38, CD105, and CD13, while the expression of CD117, CD10, CD44,and CD200 varied (FIG. 8). The average expression of each marker forumbilical cord-derived cells was the following: 62.50%±53.03% (n=2,SEM=37.50) of the cells expressed HLA-G; 25.67%±11.28% (n=5, SEM=5.04)of the cells expressed CD33; 44.45%±62.85% (n=2, SEM=44.45) of the cellsexpressed CD117; 8.33%±11.79% (n=2, SEM=8.33) of the cells expressedCD10; 21.43%±30.30% (n=2, SEM=21.43) of the cells expressed CD44; 0.0%(n=1) of the cells expressed CD200; 81.25% (n=1) of the cells expressedCD90; 64.29% (n=1) of the cells expressed CD38; 6.25% (n=1) of the cellsexpressed CD105; and 50.0% (n=1) of the cells expressed CD13.

A summary of all marker expression averages is shown in FIG. 9.

6.2.2.8 BD FACS Aria Sort Report

The three distinct populations of placental cells that expressed thegreatest percentages of HLA ABC, CD45, CD34, and CD133 (cells derivedfrom perfusate, amnion and chorion) were stained with 7AAD and theantibodies for these markers. The three populations were positivelysorted for live cells expressing the phenotype of interest. The resultsof the BD FACS Aria sort are listed in table 2.

TABLE 2 BD FACS Aria Sort Report Events Sorted (Phenotype of Cell SourceEvents Processed Interest) % Of Total Perfusate 135540110 51215 0.037786Amnion 7385933 4019 0.054414 Chorion 108498122 4016 0.003701

The three distinct populations of positively sorted cells (“sorted”) andtheir corresponding non-sorted cells were plated and the results of theculture were assessed on day 12 (Table 3). Sorted perfusate-derivedcells, plated at a cell density of 40,600/cm², resulted in small, round,non-adherent cells. Two out of the three sets of non-sortedperfusate-derived cells, each plated at a cell density of 40,600/cm²,resulted in mostly small, round, non-adherent cells with severaladherent cells located around the periphery of well. Non-sortedperfusate-derived cells, plated at a cell density of 93,800/cm²,resulted in mostly small, round, non-adherent cells with severaladherent cells located around the well peripheries.

Sorted amnion-derived cells, plated at a cell density of 6,300/cm²,resulted in small, round, non-adherent cells. Non-sorted amnion-derivedcells, plated at a cell density of 6,300/cm², resulted in small, round,non-adherent cells. Non-sorted amnion-derived cells plated at a celldensity of 62,500/cm² resulted in small, round, non-adherent cells.

Sorted chorion-derived cells, plated at a cell density of 6,300/cm²,resulted in small, round, non-adherent cells. Non-sorted chorion-derivedcells, plated at a cell density of 6,300/cm², resulted in small, round,non-adherent cells. Non-sorted chorion-derived cells plated at a celldensity of 62,500/cm², resulted in small, round, non-adherent cells.

The populations of placental stem cells described above, upon culture ontissue culture plastic, adhered to the surface and assumed acharacteristic fibroblastoid shape.

6.3 Example 3: Collection of Placental Stem Cells by Closed-CircuitPerfusion

This Example demonstrates one method of collecting placental stem cellsby perfusion.

A post-partum placenta is obtained within 24 hours after birth. Theumbilical cord is clamped with an umbilical cord clamp approximately 3to 4 inches about the placental disk, and the cord is cut above theclamp. The umbilical cord is either discarded, or processed to recover,e.g., umbilical cord stem cells, and/or to process the umbilical cordmembrane for the production of a biomaterial. Excess amniotic membraneand chorion is cut from the placenta, leaving approximately ¼ incharound the edge of the placenta. The trimmed material is discarded.

Starting from the edge of the placental membrane, the amniotic membraneis separated from the chorion using blunt dissection with the fingers.When the amniotic membrane is entirely separated from the chorion, theamniotic membrane is cut around the base of the umbilical cord withscissors, and detached from the placental disk. The amniotic membranecan be discarded, or processed, e.g., to obtain stem cells by enzymaticdigestion, or to produce, e.g., an amniotic membrane biomaterial.

The fetal side of the remaining placental material is cleaned of allvisible blood clots and residual blood using sterile gauze, and is thensterilized by wiping with an iodine swab than with an alcohol swab. Theumbilical cord is then clamped crosswise with a sterile hemostat beneaththe umbilical cord clamp, and the hemostat is rotated away, pulling thecord over the clamp to create a fold. The cord is then partially cutbelow the hemostat to expose a cross-section of the cord supported bythe clamp. Alternatively, the cord is clamped with a sterile hemostat.The cord is then placed on sterile gauze and held with the hemostat toprovide tension. The cord is then cut straight across directly below thehemostat, and the edge of the cord near the vessel is re-clamped.

The vessels exposed as described above, usually a vein and two arteries,are identified, and opened as follows. A closed alligator clamp isadvanced through the cut end of each vessel, taking care not to puncturethe clamp through the vessel wall. Insertion is halted when the tip ofthe clamp is slightly above the base of the umbilical cord. The clamp isthen slightly opened, and slowly withdrawn from the vessel to dilate thevessel.

Plastic tubing, connected to a perfusion device or peristaltic pump, isinserted into each of the placental arteries. Plastic tubing, connectedto a 250 mL collection bag, is inserted into the placental vein. Thetubing is taped into place.

A small volume of sterile injection grade 0.9% NaCl solution to checkfor leaks. If no leaks are present, the pump speed is increased, andabout 750 mL of the injection grade 0.9% NaCl solution is pumped throughthe placental vasculature. Perfusion can be aided by gently massagingthe placental disk from the outer edges to the cord. When a collectionbag is full, the bag is removed from the coupler connecting the tubingto the bag, and a new bag is connected to the tube.

When collection is finished, the collection bags are weighed andbalanced for centrifugation. After centrifugation, each bag is placedinside a plasma extractor without disturbing the pellet of cells. Thesupernatant within the bags is then removed and discarded. The bag isthen gently massaged to resuspend the cells in the remainingsupernatant. Using a sterile 1 mL syringe, about 300-500 μL of cells iswithdrawn from the collection bag, via a sampling site coupler, andtransferred to a 1.5 mL centrifuge tube. The weight and volume of theremaining perfusate are determined, and ⅓ volume of hetastarch is addedto the perfusate and mixed thoroughly. The number of cells per mL isdetermined. Red blood cells are removed from the perfusate using aplasma extractor.

Placental cells are then immediately cultured to isolate placental stemcells, or are cryopreserved for later use.

6.4 Example 4: Differentiation of Placental Stem Cells 6.4.1 Inductionof Differentiation into Neurons

Neuronal differentiation of placental stem cells can also beaccomplished as follows:

-   -   1. Placental stem cells are grown for 24 hr in preinduction        medium consisting of DMEM/20% FBS and 1 mM beta-mercaptoethanol.    -   2. The preinduction medium is removed and cells are washed with        PBS.    -   3. Neuronal induction medium consisting of DMEM and 1-10 mM        betamercaptoethanol is added to the cells. Alternatively,        induction media consisting of DMEM/2% DMSO/200 μM butylated        hydroxyanisole may be used.    -   4. In certain embodiments, morphologic and molecular changes may        occur as early as 60 minutes after exposure to serum-free media        and betamercaptoethanol. RT/PCR may be used to assess the        expression of e.g., nerve growth factor receptor and        neurofilament heavy chain genes.

6.4.2 Induction of Differentiation into Adipocytes

Several cultures of placental stem cells derived from enzymaticdigestion of amnion, at 50-70% confluency, were induced in mediumcomprising (1) DMEM/MCDB-201 with 2% FCS, 0.5% hydrocortisone, 0.5 mMisobutylmethylxanthine, 60 μM indomethacin; or (2) DMEM/MCDB-201 with 2%FCS and 0.5% linoleic acid. Cells were examined for morphologicalchanges; after 3-7 days, oil droplets appeared. Differentiation was alsoassessed by quantitative real-time PCR to examine the expression ofspecific genes associated with adipogenesis, i.e., PPAR-γ2, aP-2,lipoprotein lipase, and osteopontin. Two cultures of placental stemcells showed an increase of 6.5-fold and 24.3-fold in the expression ofadipocyte-specific genes, respectively. Four other cultures showed amoderate increase (1.5-2.0-fold) in the expression of PPAR-γ2 afterinduction of adipogenesis.

In another experiment, placental stem cells obtained from perfusate werecultured in DMEM/MCDB-201 (Chick fibroblast basal medium) with 2% FCS.The cells were trypsinized and centrifuged. The cells were resuspendedin adipo-induction medium (AIM) 1 or 2. AIM1 comprised MesenCult BasalMedium for human Mesenchymal Stem Cells (StemCell Technologies)supplemented with Mesenchymal Stem Cell Adipogenic Supplements (StemCellTechnologies). AIM2 comprised DMEM/MCDB-201 with 2% FCS and LA-BSA (1%).About 1.25×10⁵ placental stem cells were grown in 5 mL AIM1 or AIM2 inT-25 flasks. The cells were cultured in incubators for 7-21 days. Thecells developed oil droplet vacuoles in the cytoplasm, as confirmed byoil-red staining, suggesting the differentiation of the stem cells intoadipocytes.

Adipogenic differentiation of placental stem cells can also beaccomplished as follows:

-   -   1. Placental stem cells are grown in MSCGM (Cambrex) or DMEM        supplemented with 15% cord blood serum.    -   2. Three cycles of induction/maintenance are used. Each cycle        consists of feeding the placental stem cells with Adipogenesis        Induction Medium (Cambrex) and culturing the cells for 3 days        (at 37° C., 5% CO₂), followed by 1-3 days of culture in        Adipogenesis Maintenance Medium (Cambrex). An alternate        induction medium that can be used contains 1 μM dexamethasone,        0.2 mM indomethacin, 0.01 mg/ml insulin, 0.5 mM IBMX, DMEM-high        glucose, FBS, and antibiotics.    -   3. After 3 complete cycles of induction/maintenance, the cells        are cultured for an additional 7 days in adipogenesis        maintenance medium, replacing the medium every 2-3 days.    -   4. A hallmark of adipogenesis is the development of multiple        intracytoplasmic lipid vesicles that can be easily observed        using the lipophilic stain oil red 0. Expression of lipase        and/or fatty acid binding protein genes is confirmed by RT/PCR        in placental stem cells that have begun to differentiate into        adipocytes.

6.4.3 Induction of Differentiation into Osteogenic Cells

Osteogenic medium was prepared from 185 mL Cambrex Differentiation BasalMedium—Osteogenic and SingleQuots (one each of dexamethasone,1-glutamine, ascorbate, pen/strep, MCGS, and β-glycerophosphate).Placental stem cells from perfusate were plated, at about 3×10³ cellsper cm² of tissue culture surface area in 0.2-0.3 mL MSCGM per cm²tissue culture area. Typically, all cells adhered to the culture surfacefor 4-24 hours in MSCGM at 37° C. in 5% CO₂. Osteogenic differentiationwas induced by replacing the medium with Osteogenic Differentiationmedium. Cell morphology began to change from the typical spindle-shapedappearance of the adherent placental stem cells, to a cuboidalappearance, accompanied by mineralization. Some cells delaminated fromthe tissue culture surface during differentiation.

Osteogenic differentiation can also be accomplished as follows:

-   -   1. Adherent cultures of placental stem cells are cultured in        MSCGM (Cambrex) or DMEM supplemented with 15% cord blood serum.    -   2. Cultures are cultured for 24 hours in tissue culture flasks.    -   3. Osteogenic differentiation is induced by replacing MSCGM with        Osteogenic Induction Medium (Cambrex) containing 0.1 μM        dexamethasone, 0.05 mM ascorbic acid-2-phosphate, 10 mM beta        glycerophosphate.    -   4. Cells are fed every 3-4 days for 2-3 weeks with Osteogenic        Induction Medium.    -   5. Differentiation is assayed using a calcium-specific stain and        RT/PCR for alkaline phosphatase and osteopontin gene expression.

6.4.4 Induction of Differentiation into Pancreatic Cells

Pancreatic differentiation is accomplished as follows:

-   -   1. Placental stem cells are cultured in DMEM/20% CBS,        supplemented with basic fibroblast growth factor, 10 ng/ml; and        transforming growth factor beta-1, 2 ng/ml. KnockOut Serum        Replacement may be used in lieu of CBS.    -   2. Conditioned media from nestin-positive neuronal cell cultures        is added to media at a 50/50 concentration.    -   3. Cells are cultured for 14-28 days, refeeding every 3-4 days.    -   4. Differentiation is characterized by assaying for insulin        protein or insulin gene expression by RT/PCR.

6.4.5 Induction of Differentiation into Cardiac Cells

Myogenic (cardiogenic) differentiation is accomplished as follows:

-   -   1. Placental stem cells are cultured in DMEM/20% CBS,        supplemented with retinoic acid, 1 μM; basic fibroblast growth        factor, 10 ng/ml; and transforming growth factor beta-1, 2        ng/ml; and epidermal growth factor, 100 ng/ml. KnockOut Serum        Replacement (Invitrogen, Carlsbad, Calif.) may be used in lieu        of CBS.    -   2. Alternatively, placental stem cells are cultured in DMEM/20%        CBS supplemented with 50 ng/ml Cardiotropin-1 for 24 hours.    -   3. Alternatively, placental stem cells are maintained in        protein-free media for 5-7 days, then stimulated with human        myocardium extract (escalating dose analysis). Myocardium        extract is produced by homogenizing 1 μm human myocardium in 1%        HEPES buffer supplemented with 1% cord blood serum. The        suspension is incubated for 60 minutes, then centrifuged and the        supernatant collected.    -   4. Cells are cultured for 10-14 days, refeeding every 3-4 days.    -   5. Differentiation is confirmed by demonstration of cardiac        actin gene expression by RT/PCR.

6.4.6 Induction of Differentiation into Chondrocytes 6.4.6.1 GeneralMethod

Chondrogenic differentiation of placental stem cells is generallyaccomplished as follows:

-   -   1. Placental stem cells are maintained in MSCGM (Cambrex) or        DMEM supplemented with 15% cord blood serum.    -   2. Placental stem cells are aliquoted into a sterile        polypropylene tube. The cells are centrifuged (150×g for 5        minutes), and washed twice in Incomplete Chondrogenesis Medium        (Cambrex).    -   3. After the last wash, the cells are resuspended in Complete        Chondrogenesis Medium (Cambrex) containing 0.01 μg/ml TGF-beta-3        at a concentration of 5×10(5) cells/ml.    -   4. 0.5 ml of cells is aliquoted into a 15 ml polypropylene        culture tube. The cells are pelleted at 150×g for 5 minutes. The        pellet is left intact in the medium.    -   5. Loosely capped tubes are incubated at 37° C., 5% CO² for 24        hours.    -   6. The cell pellets are fed every 2-3 days with freshly prepared        complete chondrogenesis medium.    -   7. Pellets are maintained suspended in medium by daily agitation        using a low speed vortex.    -   8. Chondrogenic cell pellets are harvested after 14-28 days in        culture.    -   9. Chondrogenesis is characterized by e.g., observation of        production of esoinophilic ground substance, assessing cell        morphology, an/or RT/PCR confirmation of collagen 2 and/or        collagen 9 gene expression and/or the production of cartilage        matrix acid mucopolysaccharides, as confirmed by Alcian blue        cytochemical staining.

6.4.6.2 Differentiation of Placental and Umbilical Cord Stem Cells intoChondrogenic Cells

The Example demonstrates the differentiation of placental stem cellsinto chondrogenic cells and the development of cartilage-like tissuefrom such cells.

Cartilage is an avascular, alymphatic tissue that lacks a nerve supply.Cartilage has a low chondrocyte density (<5%), however these cells aresurprisingly efficient at maintaining the extracellular matrix aroundthem. Three main types of cartilage exist in the body: (1) articularcartilage, which facilitates joint lubrication in joints; (2)fibrocartilage, which provides shock absorption in, e.g., meniscus andintervertebral disc; and (3) elastic cartilage, which providesanatomical structure in, e.g., nose and ears. All three types ofcartilage are similar in biochemical structure.

Joint pain is a major cause of disability and provides an unmet need ofrelief in the area of orthopedics. Primary osteoarthritis (which cancause joint degeneration), and trauma are two common causes of pain.Approximately 9% of the U.S. population has osteoarthritis of hip orknee, and more than 2 million knee surgeries are performed yearly.Unfortunately, current treatments are more geared towards treatment ofsymptoms rather than repairing the cartilage. Natural repair occurs whenfibroblast-like cells invade the area and fill it with fibrous tissuewhich is neither as resilient or elastic as the normal tissue, hencecausing more damage. Treatment options historically included tissuegrafts, subchondral drilling, or total joint replacement. More recenttreatments however include CARTICEL®, an autologous chondrocyteinjection; SYNVISC® and ORTHOVISC®, which are hyaluronic acid injectionsfor temporary pain relief; and CHONDROGEN™, an injection of adultmesenchymal stem cells for meniscus repair. In general, the trend seemsto be lying more towards cellular therapies and/or tissue engineeredproducts involving chondrocytes or stem cells.

Materials and Methods.

Two placental stem cell lines, designated AC61665, P3 (passage 3) andAC63919, P5, and two umbilical cord stem cell lines, designated UC67249,P2 and UC67477, P3 were used in the studies outlined below. Humanmesenchymal stem cells (MSC) were used as positive controls, and anosteosarcoma cell line, MC3T3, and human dermal fibroblasts (HDF) wereused as negative controls.

Placental and umbilical cord stem cells were isolated and purified fromfull term human placenta by enzymatic digestion. Human MSC cells and HDFcells were purchased from Cambrex, and MC3T3 cells were purchased fromAmerican Type Culture Collection. All cell lines used were centrifugedinto pellets in polypropylene centrifuge tubes at 800 RPM for 5 minutesand grown in both chondrogenic induction media (Cambrex) andnon-inducing basal MSC media (Cambrex). Pellets were harvested andhistologically analyzed at 7, 14, 21 and 28 days by staining forglycosaminoglycans (GAGs) with Alcian Blue, and/or for collagens withSirius Red. Collagen type was further assessed with immunostaining. RNAanalysis for cartilage-specific genes was performed at 7 and 14 days.

Results

Experiment 1: Chondrogenesis studies were designed to achieve three mainobjectives: (1) to demonstrate that placental and umbilical cord stemcells can differentiate and form cartilage tissue; (2) to demonstratethat placental and umbilical cord stem cells can differentiatefunctionally into chondrocytes; and (3) to validate results obtainedwith the stem cells by evaluating control cell lines.

For objective 1, in a preliminary study, one placental stem cell linewas cultured in chondrogenic induction medium in the form of cellpellets, either with or without bone morphogenic protein (BMP) at afinal concentration of 500 ng/mL. Pellets were assessed for evidence ofchondrogenic induction every week for 4 weeks. Results indicated thatthe pellets do increase in size over time. However, no visualdifferences were noted between the BMP⁺ and BMP⁻ samples. Pellets werealso histologically analyzed for GAG's, an indicator of cartilagetissue, by staining with Alcian Blue. BMP⁺ cells generally appeared moremetabolically active with pale vacuoles whereas BMP⁻ cells were smallerwith dense-stained nuclei and less cytoplasm (reflects low metabolicactivity). At 7 days, BMP⁺ cells had stained heavily blue, while BMP⁻had stained only faintly. By 28 days of induction, both BMP⁺ and BMP⁻cells were roughly equivalently stained with Alcian Blue. Overall, celldensity decreased over time, and matrix overtook the pellet. Incontrast, the MC3T3 negative cell line did not demonstrate any presenceof GAG when stained with Alcian Blue.

Experiment 2: Based on the results of Experiment 1, a more detailedstudy was designed to assess the chondrogenic differentiation potentialof two placental stem cell and two umbilical cord stem cell lines. Inaddition to the Alcian Blue histology, cells were also stained withSirius Red, which is specific for type II collagen. Multiple pelletswere made for each cell line, with and without induction media.

The pelleted, cultured cell lines were first assessed by grossobservation for macroscopic generation of cartilage. Overall, the stemcell lines were observed to make pellets as early as day 1. Thesepellets grew over time and formed a tough matrix, appearing white,shining and cartilage-like, and became mechanically tough. By visualinspection, pellets from placental stem cells or umbilical cord stemcells were much larger than the MSC controls. Control pellets innon-induction media started to fall apart by Day 11, and were muchsmaller at 28 days than pellets developed by cells cultured inchondrogenic induction medium. Visually, there were no differencesbetween pellets formed by placental stem cells or umbilical cord.However, the UC67249 stem cell line, which was initiated indexamethasone-free media, formed larger pellets. Negative control MC3T3cells did not form pellets; however, HDFs did form pellets.

Representative pellets from all test groups were then subjected tohistological analysis for GAG's and collagen. Generally, pellets formedby the stem cells under inducing conditions were much larger and stayedintact better than pellets formed under non-inducing conditions. Pelletsformed under inducing conditions showed production of GAGs andincreasing collagen content over time, and as early as seven days, whilepellets formed under non-inducing conditions showed little to nocollagen production, as evidenced by weak Alcian Blue staining. Ingeneral, the placental stem cells and umbilical cord stem cellsappeared, by visual inspection, to produce tougher, larger pellets, andappeared to be producing more collagen over time, than the hMSCs.Moreover, over the course of the study, the collagen appeared tothicken, and the collagen type appeared to change, as evidenced bychanges in the fiber colors under polarized light (colors correlate tofiber thickness which may be indicative of collagen type). Non-inducedplacental stem cells produced much less type II collagen, if any,compared to the induced stem cells. Over the 28-day period, cell densitydecreased as matrix production increased, a characteristic of cartilagetissue.

These studies confirm that placental and umbilical cord stem cells canbe differentiated along a chondrogenic pathway, and can easily beinduced to form cartilage tissue. Initial observations indicate thatsuch stem cells are preferable to MSCs for the formation of cartilagetissue.

6.5 Example 5: Hanging Drop Culture of Placental Stem Cells

Placental adherent stem cells in culture are trypsinized at 37° C. forabout 5 minutes, and loosened from the culture dish by tapping. 10% FBSis added to the culture to stop trypsinization. The cells are diluted toabout 1×10⁴ cells per mL in about 5 mL of medium. Drops (either a singledrop or drops from a multi-channel micropipette are placed on the insideof the lid of a 100 mL Petri dish. The lid is carefully inverted andplaced on top of the bottom of the dish, which contains about 25 ml ofsterile PBS to maintain the moisture content in the dish atmosphere.Cells are grown for 6-7 days.

6.6 Example 6: Placental Tissue Digestion to Obtain Placental Stem Cells

This Example demonstrates a scaled up isolation of placental stem cellsby enzymatic digestion.

Approximately 10 grams of placental tissue (amnion and chorion) isobtained, macerated, and digested using equal volumes of collagenase A(1 mg/ml) (Sigma) and Trypsin-EDTA (0.25%) (Gibco-BRL) in a total volumeof about 30 ml for about 30 minutes at 37° C. Cells liberated by thedigestion are washed 3× with culture medium, distributed into four T-225flasks and cultured as described in Example 1. Placental stem cell yieldis between about 4×10⁸ and 5×10⁸ cells per 10 g starting material.Cells, characterized at passage 3, are predominantly CD10⁺, CD90⁺,CD105⁺, CD200⁺, CD34⁻ and CD45⁻.

6.7 Example 7: Production of Cryopreserved Stem Cell Product and StemCell Bank

This Example demonstrates the isolation of placental stem cell and theproduction of a frozen stem cell-based product.

Summary: Placental tissue is dissected and digested, followed by primaryand expansion cultures to achieve an expanded cell product that producesmany cell doses. Cells are stored in a two-tiered cell bank and aredistributed as a frozen cell product. All cell doses derived from asingle donor placenta are defined as a lot, and one placenta lot isprocessed at a time using sterile technique in a dedicated room andClass 100 laminar flow hood. The cell product is defined as beingCD105⁺, CD200⁺, CD10⁺, and CD34⁻, having a normal karyotype and nomaternal cell content.

6.7.1 Obtaining Stem Cells

Tissue Dissection and Digestion: A placenta is obtained less than 24hours after expulsion. Placental tissue is obtained from amnion, acombination of amnion and chorion, or chorion. The tissue is minced intosmall pieces, about 1 mm in size. Minced tissue is digested in 1 mg/mlCollagenase 1A for 1 hour at 37° C. followed by Trypsin-EDTA for 30minutes at 37° C. After three washes in 5% FBS in PBS, the tissue isresuspended in culture medium.

Primary Culture: The purpose of primary culture is to establish cellsfrom digested placental tissue. The digested tissue is suspended inculture medium and placed into Corning T-flasks, which are incubated ina humidified chamber maintained at 37° C. with 5% CO₂. Half of themedium is replenished after 5 days of culture. High-density colonies ofcells form by 2 weeks of culture. Colonies are harvested withTrypsin-EDTA, which is then quenched with 2% FBS in PBS. Cells arecentrifuged and resuspended in culture medium for seeding expansioncultures. These cells are defined as Passage 0 cells having doubled 0times.

Expansion Culture: Cells harvested from primary culture, harvested fromexpansion culture, or thawed from the cell bank are used to seedexpansion cultures. Cell Factories (NUNC™) are treated with 5% CO₂ inair at 50 ml/min/tray for 10 min through a sterile filter and warmed ina humidified incubator maintained at 37° C. with 5% CO₂. Cell seeds arecounted on a hemacytometer with trypan blue, and cell number, viability,passage number, and the cumulative number of doublings are recorded.Cells are suspended in culture medium to about 2.3×10⁴ cells/ml and 110ml/tray are seeded in the Cell Factories. After 3-4 days and again at5-6 days of culture, culture medium is removed and replaced with freshmedium, followed by another treatment with 5% CO₂ in air. When cellsreach approximately 10⁵ cells/cm², cells are harvested withTrypsin-EDTA, followed by quenching with 2% FBS in PBS. Cell are thencentrifuged and resuspended in culture medium.

Cryopreservation: Cells to be frozen down are harvested from culturewith Trypsin-EDTA, quenched with 2% FBS in PBS, and counted on ahemacytometer. After centrifugation, cells are resuspended with 10% DMSOin FBS to a concentration of about 1 million cells/ml for cells to beused for assembly of a cell bank, and 10 million cells/ml for individualfrozen cell doses. The cell solution is transferred to a freezingcontainer, which is placed in an isopropyl alcohol bath in a −80° C.freezer. The following day, cells are transferred to liquid nitrogen.

6.7.2 Design of a Stem Cell Bank

A “lot” is defined as all cell doses derived from a single donorplacenta. Cells maintained normal growth, karyotype, and cell surfacemaker phenotype for over 8 passages and 30 doublings during expansionculture. Given this limitation, doses comprise cells from 5 passages andabout 20 doublings. To generate a supply of equivalent cells, a singlelot is expanded in culture and is stored in a two-tiered cell bank andfrozen doses. In particular, cells harvested from the primary culture,which are defined as Passage 0 cells having undergone 0 doublings, areused to initiate an expansion culture. After the first passage,approximately 4 doublings occur, and cells are frozen in a Master CellBank (MCB). Vials from the MCB are used to seed additional expansioncultures. After two additional passages of cells thawed from the MCB,cells are frozen down in a Working Cell Bank (WCB), approximately 12cumulative doublings. Vials from the WCB are used to seed an expansionculture for another 2 passages, resulting in Passage 5 cells atapproximately 20 doublings that are frozen down into individual doses.

6.7.3 Thawing Cells for Culture

Frozen containers of cells are placed into a sealed plastic bag andimmersed in a 37° C. water bath. Containers are gently swirled until allof the contents are melted except for a small piece of ice. Containersare removed from the sealed plastic bag and a 10× volume of culturemedium is slowly added to the cells with gentle mixing. A sample iscounted on the hemacytometer and seeded into expansion cultures.

6.7.4 Thawing Cells for Injection

Frozen containers of cells are transferred to the administration site ina dry nitrogen shipper. Prior to administration, containers are placedinto a sealed plastic bag and immersed in a 37° C. water bath.Containers are gently swirled until all of the contents are meltedexcept for a small piece of ice. Containers are removed from the sealedplastic bag and an equal volume of 2.5% HSA/5% Dextran is added. Cellsare injected with no further washing.

6.7.5 Testing and Specifications

A maternal blood sample accompanies all donor placentas. The sample isscreened for Hepatitis B core antibody and surface antigen, Hepatitis CVirus antibody and nucleic acid, and HIV I and II antibody and nucleicacid. Placental processing and primary culture begins prior to thereceipt of test results, but continues only for placentas associatedwith maternal blood samples testing negative for all viruses. A lot isrejected if the donor tests positive for any pathogen. In addition, thetests described in Table 3 are performed on the MCB, the WCB, and asample of the cell dose material derived from a vial of the WCB. A lotis released only when all specifications are met.

TABLE 3 Cell testing and specifications Test Methods Required ResultSterility BD BACTEC PEDS Negative PLUS/F and BACTEC Myco/F LyticEndotoxin LAL gel clot ≤5 EU/ml* Viability Trypan Blue >70% viableMycoplasma Direct culture, DNA- Negative fluorochrome (FDA PTC 1993)Identity Flow cytometry (see CD105⁺, CD200⁺, CD10⁺, CD34⁻ below) CellPurity Microsatellite No contaminating cell detected Karyotype G-bandingand Normal chromosome count on metaphase cells *For the product designedto be 40 ml of frozen cells/dose and a maximum of 5 EU/ml, the cellproduct is below the upper limit of 5 EU/kg/dose for recipients over 40kg in body weight.

6.7.6 Surface Marker Phenotype Analysis

Cells are placed in 1% paraformaldehyde (PFA) in PBS for 20 minutes andstored in a refrigerator until stained (up to a week). Cells are washedwith 2% FBS, 0.05% sodium azide in PBS (Staining Buffer) and thenresuspended in staining buffer. Cells are stained with the followingantibody conjugates: CD105-FITC, CD200-PE, CD34-PECy7, CD10-APC. Cellsare also stained with isotype controls. After 30 minute incubation, thecells are washed and resuspended with Staining Buffer, followed byanalysis on a flow cytometer. Cells having an increased fluorescencecompared to isotype controls are counted as positive for a marker.

6.8 Example 8: Identification of Placental Stem Cell-Specific Genes

Gene expression patterns from placental stem cells from amnion-chorion(AC) and umbilical cord (UC) were compared to gene expression patternsof multipotent bone marrow-derived mesenchymal stem cells (BM) anddermal fibroblasts (DF), the latter of which is considered to beterminally differentiated. Cells were grown for a single passage, anintermediate number of passages, and large number of passages (includinguntil senescence). Results indicate that the number of populationdoublings has a major impact on gene expression. A set of genes wasidentified that are up-regulated in AC and UC, and either down-regulatedor absent in BM and DF, and that are expressed independent of passagenumber. This set of placental stem cell- or umbilical cord stemcell-specific genes encodes a number of cytoskeleton and cell-to-celladhesion proteins associated with epithelial cells and animmunoglobulin-like surface protein, CD200, implicated in maternal-fetalimmune tolerance. Placental stem cells and umbilical cord stem cellswill be referred to collectively hereinafter in this Example as AC/UCstem cells.

6.8.1 Methods and Materials 6.8.1.1 Cells and Cell Culture

BM (Cat# PT-2501) and DF (Cat# CC-2511) were purchased from Cambrex. ACand UC originated from passage 0 tissue culture flasks. AC and UC in theflasks were obtained by digestion from a donor placenta designated2063919. T-75 culture flasks were seeded at 6000 cells/cm² and cellswere passaged when they became confluent. Population doublings wereestimated from trypan blue cell counts. Cultures were assayed for geneexpression after 3, 11-14, and 24-38 population doublings.

6.8.1.2 RNA, Microarrays, and Analysis

Cells were lysed directly in their tissue culture flasks, with theexception of one culture that was trypsinized prior to lysis. Total RNAwas isolated with the RNeasy kit from QIAGEN. RNA integrity andconcentrations were determined with an Agilent 2100 Bioanalyzer. Tenmicrograms of total RNA from each culture were hybridized on anAffymetrix GENECHIP® platform. Total RNA was converted to labeled cRNAsand hybridized to oligonucleotide Human Genome U133A 2.0 arraysaccording to the manufacture's methods. Image files were processed withthe Affymetrix MAS 5.0 software, and normalized and analyzed withAgilent GeneSpring 7.3 software.

6.8.2 Results 6.8.2.1 Selection of BM-MSC, AC/UC Stem Cell, and DFCulture Time-Points for Microarray Analyses

To establish a gene expression pattern unique to AC/UC stem cells, twostem cell lines, AC(6) and UC(6), were cultured in parallel with BM-MSCand DF. To maximize identifying a gene expression profile attributableto cellular origin and minimize exogenous influences all cells weregrown in the same medium, seeded, and sub-cultured using the samecriteria. Cells were harvested after 3 population doublings, 11-14doublings, or 35 doublings or senescence, whichever came first. Geneswhose expression in AC/UC stem cells are unchanged by time-in-cultureand are up-regulated relative to BM and DF are candidates for AC/UC stemcell-specific genes.

FIG. 10 shows growth profiles for the four cell lines in the study;circles indicate which cultures were harvested for RNA isolation. Intotal twelve samples were collected. BM, AC(6), and UC(6) were harvestedafter three population doublings; these samples were regarded as beingin culture for a “short” period of time. A short-term DF sample was notcollected. Intermediate length cultures, 11 to 14 doublings, werecollected for all cell types. Long-term cultures were collected from allcell lines at about 35 population doublings or just prior to senescence,whichever came first. Senescence occurred before 15 doublings for BM andat 25 doublings for DF. The purchased BM and DF cells were expanded manytimes prior to gene analysis, and cannot be considered early-stage.However, operationally, BM grown for three doublings (BM-03) are deemeda short-term culture. Likewise, BM-11 is operationally referred to as anintermediate length culture, but because senescence occurred at 14doublings, BM-11 is most likely a long-term culture biologically.

6.8.2.2 Hierarchical Clustering Shows Relatedness Between BM, AC/UC StemCells, and DF

Microarray analysis identifies patterns of gene expression, andhierarchical clustering (HC) attempts to find similarities in thecontext of two dimensions—genes in the first dimension and differentconditions (different RNA samples) in the second. The GeneChips used inthis experiment contained over 22,000 probe sets (referred to as the“all genes list”), but many of these sets interrogate genes that are notexpressed in any condition. To reduce the all genes list, genes notexpressed or expressed at low levels (raw values below 250) in allsamples were eliminated to yield a list of 8,215 genes.

6.8.2.3 Gene Expression Analysis Using the Line Graph View

Gene expression patterns of the 8215 genes were displayed using the linegraph view in GeneSpring (FIG. 11). The x-axis shows the twelveexperimental conditions and the y-axis shows the normalized probe setexpression values on a log scale. The y-axis covers a 10,000-fold range,and genes that are not expressed or expressed at very low levels are setto a value of 0.01. By default the normalized value is set to 1. Eachline represents a single gene (actually a probe set, some genes havemultiple probe sets) and runs across all twelve conditions as a singlecolor. Colors depict relative expression levels, as described for theheatmaps, but the coloring pattern is determined by selecting onecondition. AC-03 is the selected condition in FIG. 11. Genesup-regulated relative to the normalized value are displayed by thesoftware as red, and those that are down-regulated, are displayed asblue. The obvious upward and downward pointing spikes in AC-03 throughUC-11 indicate that many genes are differentially expressed across theseconditions. The striking similarity in the color patterns between AC-03and UC-03 show that many of the same genes are up or down-regulated inthese two samples. Horizontal line segments indicate that a gene'sexpression level is unchanged across a number of conditions. This ismost notable by comparing UC-36, UC-38, and UC-38-T. There are noobvious spikes, but there is a subtle trend in that a number of redlines between UC-36 and UC-38-T are below the normalized value of 1.This indicates that these genes, which are up-regulated in AC-03 andUC-03, are down-regulated in the later cultures. The fact that theexpression patterns between UC-38 and UC-38-T are so similar indicatesthat trypsinizing cells just prior to RNA isolation has little effect ongene expression.

In addition to the computationally intensive HC method, by visualinspection the two BM samples are more similar to each other than to theother conditions. The same is true for the two DF cultures. And despitethe large number of differentially expressed genes present in the BM andDF samples, the general appearance suggests that two BMs and the two DFsare more similar to each other than to AC/UC stem cells. This isconfirmed by the HC results described above.

When the above process is applied using AC-11 as the selected condition,it is clear that AC-11 and UC-11 share many of the same differentiallyexpressed genes, but the total number of genes in common between thesetwo conditions appears less than the number of differentially expressedgenes shared by AC-03 and UC-03. FIG. 12 shows genes differentiallyover-expressed, by six-fold or more relative to the baseline, in AC-03.The majority of genes up-regulated in AC-03 are also up-regulated inUC-03, and more divergent in BM and DF.

6.8.2.4 Filtering Methods Used to Identify AC/UC Stem Cell-SpecificGenes

Genes that remain constant across all AC/UC samples, and aredown-regulated in BM and DF, are considered AC/UC stem cell-specific.Two filtering methods were combined to create a list of 58 AC/UC stemcell-specific genes (Table 4).

TABLE 4 58 Placental stem cell or Umbilical cord stem cell-specificgenes Biological Process, Symbol Gene Description, and AdditionalAnnotation ACTG2 actin, gamma 2, smooth muscle development,cytoskeleton, muscle, enteric expressed in umbilical cord artery andprostate epithelia ADARB1 adenosine deaminase, RNA- RNA processing,central nervous system specific, B1 (RED1 homolog development rat)AMIGO2 amphoterin induced gene 2 homophilic and heterophilic celladhesion, adhesion molecule with lg like domain 2 ARTS-1 type 1 tumornecrosis factor proteolysis, antigen processing, receptor sheddingangiogenesis, expressed in placenta aminopeptidase regulator B4GALT6UDP-Gal: betaGlcNAc beta 1,4- carbohydrate metabolism, integral togalactosyltransferase, membrane, may function in intercellularpolypeptide 6 recognition and/or adhesion BCHE butyrylcholinesterasecholinesterase activity, serine esterase activity, hydrolase activityC11orf9 chromosome 11 open reading hypothetical protein, p53-liketranscription frame 9 factor, expressed in retinal pigment epitheliumCD200 CD200 antigen immunoglobulin-like, surface protein, inhibitsmacrophage COL4A1 collagen, type IV, alpha I ECM, basement membrane,afibrillar collagen, contains arresten domain COL4A2 collagen, type IV,alpha 2 ECM, biogenesis, basement membrane, coexpressed with COL 4A1,down-reg. in dysplastic epithelia CPA4 carboxypeptidase A4 proteolytic,histone acetylation, maternal imprinted, high expression in prostatecancer cell lines DMD dystrophin (muscular muscle contraction, cellshape and cell size dystrophy, Duchenne and control, muscle developmentBecker types) DSC3 desmocollin 3 homophilic cell-cell adhesion,localized to desmosomes DSG2 desmoglein 2 homophilic cell-cell adhesion,localized to desmosomes ELOVL2 elongation of very long chain fatty acidbiosynthesis, lipid biosynthesis fatty acids (FEN1/Elo2, SUR4/Elo3,yeast)-like 2 F2RL1 coagulation factor II (thrombin) G-protein coupledreceptor protein receptor-like 1 signaling pathway, highly expressed incolon epithelia and neuronal elements FLJ10781 hypothetical proteinFLJ10781 — GATA6 GATA binding protein 6 transcription factor, muscledevelopment GPR126 G protein-coupled receptor 126 signal transduction,neuropeptide signaling pathway GPRC5B G protein-coupled receptor,G-protein coupled receptor protein family C, group 5, member B signalingpathway, ICAM1 intercellular adhesion molecule cell-cell adhesion, celladhesion, 1 (CD54), human rhinovirus transmembrane receptor activity,receptor expressed in conjunctival epithelium IER3 immediate earlyresponse 3 anti-apoptosis, embryogenesis and morphogenesis, cell growthand/or maintenance IGFBP7 insulin-like growth factor negative regulationof cell proliferation, binding protein 7 overexpressed in senescentepithelial cells IL1A interleukin 1, alpha immune response, signaltransduction, cytokine activity, cell proliferation, differentiation,apoptosis IL1B interleukin 1, beta immune response, signal transduction,cytokine activity, cell proliferation, differentiation, apoptosis 1L6interleukin 6 (interferon, beta 2) cell surface receptor linked signaltransduction, immune response KRT18 keratin 18 morphogenesis,intermediate filament, expressed in placenta, fetal, and epithelialtissues KRT8 keratin 8 cytoskeleton organization and biogenesis,phosphorylation, intermediate filament, coexpressed with KRTIB LIPGlipase, endothelial lipid metabolism, lipoprotein lipase activity, lipidtransporter, phospholipase activity, involved in vascular biology LRAPleukocyte-derived arginine antigen processing, endogenous antigenaminopeptidase via MHC class I; N-terminal aminopeptidase activity MATN2matrilin 2 widely expressed in cell lines of fibroblastic or epithelialorigin, nonarticular cartilage ECM MEST mesoderm specific transcriptpaternally imprinted gene, development of homolog (mouse) mesodermaltissues, expressed in fetal tissues and fibroblasts NFE2L3 nuclearfactor (erythroid- transcription co-factor, highly expressed in derived2)-like 3 primary placental cytotrophoblasts but not in placentalfibroblasts NUAK1 NUAK family, SNF1-like protein amino acidphosphorylation, kinase, I protein serine-threonine kinase activityPCDH7 BH-protocadherin (brain-heart) cell-cell adhesion and recognition,containing 7 cadherin repeats PDLIM3 PDZ and LIM domain 3alpha-actinin-2-associated LIM protein, cytoskeleton protein binding,expressed in skeletal muscle PKP2 plakophilin 2 cell-cell adhesion,localized to desmosomes, found in epithelia, binds cadherins andintermediate filament RTN1 reticulon 1 signal transduction; neurondifferentiation, neuroendocrine secretion, membrane trafficking inneuroendocrine cells SERPINB9 serpin peptidase inhibitor, ciade serineprotease inhibitor, coagulation, B (ovalbumin), member 9 fibrinolysis,complement fixation, matrix remodeling, expressed in placenta ST3GAL6sialyltransferase 10 amino sugar metabolism, protein amino acidglycosylation, glycolipid metabolism, protein-lipoylation ST6GALNAC5sialyltransferase 7E protein amino acid glycosylation, gangliosidebiosynthesis SLC12A8 solute carrier family 12 amino acid-polyaminetransporter activity, (sodium/potassium/chloride cation-chloridecotransporter 9, possible transporters), member 8 role in epithelialimmunity (psoriasis) TCF21 transcription factor 21 regulation oftranscription, mesoderm development, found in epithelial cells of thekidney TGFB2 transforming growth factor, regulation of cell cycle,signal beta 2 transduction, cell-cell signaling, cell proliferation,cell growth VTN vitronectin (serum spreading immune response, celladhesion, secreted factor, somatomedin B, protein, binds ECM complementS-protein) ZC3H12A zinc finger CCCM-type MCP-I treatment-inducedprotein, nucleic containing 12A acid binding, hypothetical zinc fingerprotein

First, 58 genes were identified by selecting those genes over-expressedthree-fold in at least seven of eight AC/UC stem cell conditionsrelative to all BM and DF samples (FIG. 13). Filtering on eight of theeight AC/UC stem cell conditions yielded a similar list. The secondfiltering method used “absent” and “present” calls provided by theAffymetrix MAS 5.0 software. A list was created by identifying genesabsent in all BM and DF conditions and present in AC-03, AC-11, UC-03,and UC-11. Gene calls in the later AC/UC stem cell conditions were notstipulated.

The two lists overlapped significantly and were combined. The combinedlist was trimmed further by eliminating (1) several genes expressed atvery low levels in most or all AC/UC stem cell conditions, and (2) genescarried on the Y chromosome. AC and UC cells used in this study wereconfirmed to be male by FISH analysis, and the BM and DF were derivedfrom a female donor. The resulting list of 46 AC/UC stem cell-specificgenes is shown in Table 5.

TABLE 5 AC/UC-Specific Genes Listed by Ontology Cell Adhesion AMIGO2B4GALT6 DSC3 DSG2 ICAM1 PCDH7 PKP2 VTN Cytoskeletal ACTG2 DMD KRT18 KRT8PDLIM3 Development ADARB1 IER3 IGFBP7 IL1A IL1B MEST TGFB2 ECM COL4A1COL4A2 MATN2 VTN Implicated in Epithelia ACTG2 C11orf9 COL4A1 COL4A2DSC3 DSG2 F2RL1 ICAM1 IGFBP7 IL6 KRT18 KRT8 MATN2 PKP2 SLC12A8 TCF21Glycosylation B4GALT6 ST3GAL6 ST6GALNAC5 Transcription C11orf9? GATA6NFE2L3 TCF21 Response Immune ARTS-1 CD200 IL1A IL1B IL6 LRAP SLC12A8 VTNProteolysis ARTS-1 CPA4 LRAP Signaling F2RL1 GPR126 GPRC5B IL1A IL1B IL6RTN1 TGFB2

This list of 46 genes encodes a collection of proteins presenting anumber of ontology groups. The most highly represented group, celladhesion, contains eight genes. No genes encode proteins involved in DNAreplication or cell division. Sixteen genes with specific references toepithelia are also listed.

6.8.3 Discussion

An expression pattern specific to placental stem cells, anddistinguishable from bone marrow-derived mesenchymal cells, wasidentified. Operationally, this pattern includes 46 genes that are overexpressed in all placental stem cell samples relative to all BM and DFsamples.

The experimental design compared cells cultured for short, medium, andlong periods of time in culture. For AC and UC cells, each cultureperiod has a characteristic set of differentially expressed genes.During the short-term or early phase (AC-03 and UC-03) two hundredup-regulated genes regress to the mean after eight population doublings.Without being bound by theory, it is likely that this early stage geneexpression pattern resembles the expression profile of AC and UC whilein the natural placental environment. In the placenta these cells arenot actively dividing, they are metabolizing nutrients, signalingbetween themselves, and securing their location by remodeling theextracellular surroundings.

Gene expression by the intermediate length cultures is defined by rapidcell division and genes differentially expressed at this time are quitedifferent from those differentially expressed during the early phase.Many of the genes up-regulated in AC-11 and UC-11, along with BM-03 andDF-14, are involved in chromosome replication and cell division. Basedon gene expression, BM-03 appears biologically to be a mid-term culture.In this middle stage cell type-specific gene expression is overshadowedby cellular proliferation. In addition, almost every gene over expressedin the short-term AC or UC cultures is down-regulated in the middle andlater stage conditions. 143 genes were up-regulated five-fold duringthis highly proliferative phase, constituting approximately 1.7% of theexpressed genes.

The long-term cultures represent the final or senescent phase. In thisphase, cells have exhausted their ability to divide, and, especially forAC and UC, the absolute number of differentially expressed genes isnoticeably reduced. This may be the result of cells being fully adaptedto their culture environment and a consequently reduced burden tobiosynthesize. Surprisingly, late BM and DF cultures do not display thissame behavior; a large number of genes are differentially expressed inBM-11 and DF-24 relative to AC and UC and the normalized value of 1. ACand UC are distinguishable from BM and DF most notably in the long-termcultures.

The placental stem cell-specific gene list described here is diverse.COL4A1 and COL4A2 are coordinately regulated, and KRT18 and KRT8 alsoappear to be co-expressed. Eight of the genes encode proteins involvedin cell to cell contact, three of which (DSC3, DSG2, and PKP2) arelocalized to desmosomes, intercellular contact points anchored tointermediate filament cytoskeleton proteins such as keratin 18 andkeratin 8. Tight cell-to-cell contact is characteristic of epithelialand endothelial cells and not typically associated with fibroblasts.Table 3 lists 16 genes, of the 46 total, characteristic to epithelialcells. Placental stem cells are generally described as fibroblast-likesmall spindle-shaped cells. This morphology is typically distinct fromBM and DF, especially at lower cell densities. Also of note is theexpression pattern of CD200, which is present in AC/UC stem cell andabsent in all BM and DF samples. Moreover, CD200 has been shown to beassociated with immune tolerance in the placenta during fetaldevelopment (see, e.g., Clark et al., Am. J. Reprod. Immunol.50(3):187-195 (2003)).

This subset of genes of 46 genes constitutes a set of molecularbiomarkers that distinguishes AC/UC stem cells from bone marrow-derivedmesenchymal stem cells or fibroblasts.

6.9 Example 6.9: Differentiation of Adherent Placental Stem Cells intoOsteogenic Cells

This example describes the results of experiments demonstrating theability of placental stem cells to differenate into osteogenic cells.This example also demonstrates the ability of such osteogenic cells tomineralize, or to contribute to mineralization, of an appropriatescaffold in vitro.

6.9.1 Expression of Osteogenic Markers by Differentiated Placental StemCells

Initially, the ability of placental stem cells to differentiate intoosteogenic precursors was assessed by monitoring alkaline phosphatase(AP) activity. AP activity is a commonly used early marker for boneformation. See, e.g., Kasten et al., 2005, Biomaterials 26:5879-89.

6.9.1.1 Reagents

DMEM-LG, insulin-transferrin-selenium-G supplement (ITS),penicillin-streptomycin (P/S), PicoGreen dsDNA fluorescent assay werepurchased from Invitrogen (Eugene, Oreg.). MCDB201, linoleic acid,dexamethasone, L-ascorbic acid, and epidermal growth factor werepurchased from Sigma (St. Louis, Mo.). Fetal bovine serum (FBS) andplatelet-derived growth factor were obtained from Hyclone (Logan, Utah)and R&D Systems (Minneapolis, Minn.), respectively. Cryopreservedbone-marrow derived mesenchymal stem cells (MSC), mesenchymal stem cellgrowth medium (designated in this Example as “basal”), and osteogenicdifferentiation medium (OS) were purchased from Cambrex (EastRutherford, N.J.). See also Section 5.5.4, above.

6.9.1.2 Cell Culture

Adherent placental stem cells were isolated from the placenta by one ofseveral methods including physical disruption of tissue from severaldifferent anatomical sites within the placenta. Adherent placental stemcells were established and subcultured at 5×10³ cells/cm² in AnthrolBmedium (60% DMEM-LG, 40% MCDB201, 2% FBS, 1× P/S, 180 ng/mL linoleicacid, 0.05 μM dexamethasone, 0.1 mM L-ascorbic acid, 10 ng/mLplatelet-derived growth factor and 10 ng/mL epidermal growth factor).Bone marrow-MSC were subcultured in basal medium at 5×10³ cells/cm². Forexperimental studies on tissue culture polystyrene, placental stem cellsand/or mesenchymal stem cells were seeded in either basal or AnthrolBmedium at 5×10³ cells/cm² then maintained in either AnthrolB medium orinduced with OS for up to 5 weeks; cells were fed bi-weekly with freshmedium. For studies on 3 dimensional scaffolds, placental stem cells ina volume of 100 μl of AnthrolB medium were seeded (2.5×10⁵cells/scaffold) on calcium phosphate (CaP, BD Biosciences, San JoseCalif.) or □ β-tri-calcium phosphate (TCP, Therics, Akron, Ohio;VITOSS®, Orthovita, Inc.; Malvern, Pa.; HEALOS™II; DePuy Spine, Inc.;Raynham, Mass.) scaffolds. After 1-2 hour incubation at 37° C., thewells containing the scaffolds were supplemented with 180 μl of medium.After 3-4 days, half of the samples were maintained in AnthrolB mediumand the other half of the samples were induced with OS medium. Mediumwas exchanged on a bi-weekly basis.

6.9.1.3 Alkaline Phosphatase Assay

Alkaline phosphatase (AP) activity in cell lysates was determined usinga colorimetric assay (Cell Biolabs, San Diego, Calif.), which measuresthe formation of p-nitrophenol product; AP activity was normalized to μgof DNA (to account for any differences in cell number) using thePicoGreen dsDNA fluorescent assay (Invitrogen, Eugene, Oreg.). Toascertain AP activity of cells cultured on scaffolds, cell-scaffoldconstructs were washed with PBS, immersed in cell lysis buffer, crushedwith a pipette tip, and centrifuged at 12000 g. Supernatants were thenanalyzed for AP activity and DNA content as described above.

6.9.1.4 Results and Discussion

Placental stem cells and mesenchymal stem cells were seeded in eitherbasal medium (Cambrex) or AnthrolB medium, then maintained in eitherbasal, OS, or AnthrolB medium for 3 weeks (cells seeded in basal mediumand induced with OS medium are designated as “basal-OS” in FIG. 14). Asshown in FIGS. 14A and 14B, cells seeded and maintained in basal mediumshow the lowest AP activity, as expected, while cells seeded in basalmedium and induced with OS medium show comparatively higher levels of APactivity. Interestingly, cells seeded and maintained in AnthrolB showthe highest levels of AP activity, higher even than cells seeded inAnthrolB medium and induced with OS medium. Thus, this experimentdemonstrated that PDACs can differentiate into osteogenic precursorcells when cultured in appropriate media.

6.9.2 Functional Characterization of PDACs Differentiation intoOsteogenic Cells

This example describes the results of experiments to assess thefunctional abilities of ostoegenic cells differentiated from placentalstem cells. Specifically, the ability of the osteogenic cells to deposita mineralized matrix was assessed. Placental stem cells were preparedand cultured as described in Example 6.9.1, above, except that placentalstem cells were seeded and cultured in AnthrolB medium for 3 days, theneither maintained in AnthrolB medium or induced with OS medium for 3weeks. Mineralization was assessed by von Kossa staining, a calciumassay, and scanning electromicrograph (SEM) visualization.

6.9.2.1 Von Kossa Staining

Specimens were stained for mineral by the von Kossa method. Inparticular, cell layers were fixed with 10% formalin for 10 minutes,incubated with 5% silver nitrate under ultraviolet light for 20 minutes,washed with deionized water, incubated with 5% thiosulfate for 5minutes, and washed thoroughly with deionized water.

6.9.2.2 Calcium Assay

Cell monolayers were rinsed twice with phosphate-buffered saline (PBS)and scraped off the dish in 0.5N HCl. Accumulated calcium was extractedfrom the cellular component by incubating overnight at 4 C on an orbitalshaker, followed by centrifugation at 2000 g for 10 minutes. Thesupernatant was used for calcium determination using a calciumquantification kit from Stanbio Laboratory (Boerne, Tex.). Calciumlevels were normalized to total cell protein to account for anydifferences in cell number.

6.9.2.3 SEM Analysis

Samples for SEM were fixed in 10% formalin for 15 minutes, washed withPBS, and dehydrated in a graded series of ethanol (20, 40, 60, 80, and100%). Scaffolds were embedded in paraffin after ethanol dehydration tofacilitate sectioning. After sectioning, samples were incubated inxylene and dehydrated in a graded series of ethanol as described above.All specimens were then sputter coated with gold and analyzed using aJEOL JSM-6400F field emission SEM (Evans Analytical Group, East Windsor,N.J.).

6.9.2.4 Results and Discussion

As shown in FIG. 15A, adherent placental stem cells induced with OSmedium show evidence of calcium deposits by von Kossa staining; thesedeposits were not observed in cells maintained in AnthrolB medium. Toquantify these levels of mineralized matrix, calcium associated withcell monolayers was determined. As shown in FIG. 15B, three-fold morecalcium was recovered from cell layers induced with OS medium comparedto those cultured in AnthrolB medium. Together with the von Kossastaining data, the calcium extraction results show that placental stemcells induced with OS medium form mineralized matrix. To visualizemineralized matrix at a high resolution, samples of placental stem cellseither maintained in AnthrolB medium (FIG. 16A) or induced with OSmedium (FIG. 16B) were subjected to SEM analysis.

Deposits of matrix mineralized are clearly evident in placental stemcells induced with OS medium, while no such accumulations of mineralizeddeposits are seen in placental stem cells cultured in AnthrolB medium.Elemental mapping of deposits in FIG. 16B by X-ray analysis confirm thatthese nodules are composed of calcium and phosphate.

The apparent lack of correlation between results in FIG. 14 (increasedAP activity in the presence of AnthrolB medium) and FIGS. 15 and 16(lack of mineralization in the presence of AnthrolB medium) can beexplained by the fact that AnthrolB does not contain β-glycerophosphate,which is required as a source of phosphate for mineralization of thematrix. Dexamethasone and ascorbic acid, which are present in AnthrolBmedium as well as OS medium, are common inducers of osteogenicdifferentiation in stem cells. See, e.g., Sun et al., 2006, Biomaterials27:5651-7. β-glycerophosphate is usually included in osteogenicdifferentiation medium as a source of phosphate to enable cell-mediatedmineralization of the matrix; it is not, in general, recognized as aninducer per se of osteogenic differentiation. The AP activity datasuggests that placental stem cells seeded and maintained in AnthrolBhave the highest osteogenic differentiation potential; it is quiteprobable that mineralization was not observed in placental stem cellscultured in AnthrolB medium due to the lack of β-glycerophosphate.

6.9.3 Differentiation of PDACs on a Three Dimensional Scaffold

This example describes differentiation of placental stem cells intoosteogenic cells on a three dimensional substrate. Since calciumphosphate- and apatitite-based biomaterials have been clinically appliedfor the treatment of fractures and bone defects, two commerciallyavailable ceramic scaffolds were chosen to evaluate placental stem cellattachment and osteogenic functionality on 3 dimensional (3D) scaffolds.Placental stem cells and mesenchymal stem cells were seeded ontoscaffolds and evaluated for their ability to attach and remain adherentto the scaffolds during long-term in vitro culture. As shown in FIG. 17,placental stem cells, as well as mesenchymal stem cells, preferentiallyattach to (3-tri-calcium phosphate (TCaP) compared to calcium phosphate(CaP) scaffolds, with placental stem cells and mesenchymal stem cellsshowing similar levels of attachment to TCaP scaffolds. In addition,throughout the duration of the time course, there are consistently morecells (both placental stem cells and mesenchymal stem cells) present onTCaP versus CaP scaffolds. By the second week of culture, both adherentplacental stem cells and mesenchymal stem cells were no longerdetectable on CaP scaffolds. These data are supported by analysis ofoxygen consumption in culture medium using oxygen sensor plates.Together, these results suggest, at least under certain conditions, thatTCaP scaffolds are more preferable for maintaining PDAC viability thanCaP scaffolds.

To assess osteogenic differentiation on scaffolds, AP activity of cellscultured on scaffolds was monitored in an AP assay performed asdescribed above. Placental stem cells and mesenchymal stem cells wereseeded in AnthrolB medium then either maintained in AnthrolB medium orOS medium for the duration of the experiment. As shown in FIG. 18,placental stem cells on TCaP scaffolds show similar AP activity whethercultured in AnthrolB medium or OS medium, while MSC on TCaP scaffoldsdisplayed higher AP activity in Anthro medium than cells cultured in OSmedium. These results are consistent with AP activity data obtained on2D surfaces, namely that factors present in AnthrolB medium may bestimulating AP activity to similar levels as OS medium. For both MSCsand PDACs, no AP activity was detected in cells seeded on CaP scaffolds.

To functionally assess placental stem cell bone matrix formation on 3Dscaffolds, adherent placental stem cells seeded on scaffolds andcultured in either AnthrolB or OS medium for 3-5 weeks were subjected toSEM analysis. As shown in FIG. 19, SEM of TCaP scaffold which werecultured in the absence of cells showed a highly porous surface (denotedby an arrow) by the presence of abundant pores. Scaffolds cultured witheither placental stem cells or mesenchymal stem cells show a lack ofsurface porosity, suggesting that cells are forming a monolayerconsisting of either or both cell bilayers or extracellular matrixproteins surrounding the scaffold.

To elucidate whether mineralized bone matrix formation was occurringinside the scaffolds by cells, cross sections of the cell-TCaP scaffoldconstruct were analyzed by SEM at a high resolution (5000×). As shown inFIG. 19, scaffolds cultured in the absence of cells were characterizedby sharp edges of the TCaP crystal composing the scaffold. Howeverscaffolds seeded with placental stem cells or mesenchymal stem cellslack these sharp edges and instead are decorated with nodularstructures, closely resembling those observed in FIG. 17, suggesting theformation of mineralized bone matrix by both placental stem cells andmesenchymal stem cells on TCaP scaffolds.

6.10 Example 10: Differentiation of Placental Stem Cells into OsteogenicPrecursors

This example describes the results of experiments assessing thedifferentiation of placental stem cells isolated by perfusion intoosteogenic precursor cells. Cells were isolated from human placenta byperfusion according to Example 6.3.

Following collection, the human placental perfusate (HPP) cells werecultured in DMEM medium containing 10% FBS or OS for 10 days on VITOSS®(Orthovita, Inc.; Malvern, Pa.). Cells were pelleted by centrifugationat 1,200 rpm for 5 min. After removal of the remaining fluid from thecell pellets, cells were resuspended in 20 μl of PBS-2% fetal bovineserum at the designated cell numbers (25 k, 50 k or 100 k). Scaffoldswere then cut into 2×3×5 mm³ pieces and placed in the wells of 96-wellplates. Cell suspensions were loaded directly onto the scaffolds andincubated at 37° C. with the presence of 5% CO₂ for 30 min followed bydispensing 200 μl of Cambrex Osteogenic Differentiation medium (Cat. #PT-3002) to immerse cell-scaffolds. For cell viability assay, scaffoldsloaded with cells were transferred to the BD Oxygen Biosensor System (BDBiosciences, Cat#353830) and immersed by 200 μl of Cambrex OsteogenicDifferentiation medium.

Osteogenic potential was then evaluated by staining and by monitoring APactivity. In particular, cells were stained with alizarin red accordingto conventional techniques for the presence of calcium. As shown in FIG.20, both the stem cells and MSCs deposited a calcium-containingmineralized matrix in OS medium, but not in DMEM.

AP assays were performed after culturing in OS for ten days. To do so,HPP on scaffolds were lysed in 100 μl of PBS containing 0.2% TritonX-100 by freezing and thawing for two times. 5 μl of cell lysate wasused for measuring the alkaline phosphatase activity by using BioAssaySystems' QuantiChrom Alkaline Phosphatase Assay Kit (Cat# DALP-250) asinstructed by the vendor guideline. Results of the assays are presentedin FIG. 21, which shows that both MSCs and HPPs exhibited AP activityfollowing 10 days' culturing in OS medium. Thus, these experimentsdemonstrate that the stem cell fraction containing cells obtained asdescribed above also had the ability to differentiate into osteogenicprecursor cells.

6.11 Example 11: In Vivo Models for Bone Repair with CompositionsComprising Placental Stem Cells

This example describes experiments that are performed in order to assesstreatment of bone defects with compositions comprising placental stemcells. Several models of bone disease are adapted to assess applicationof such treatments to different bone diseases.

To model cranial bilateral defect, a defect of 3 mm×5 mm is surgicallycreated on each side of the cranium of male athymic rats. The defectsare treated with matrix only, matrix in combination with PDACs, andmatrix in combination with HPPs. The amounts of PDACs are varied toassess dose-dependency of the different treatments. Different matrixmaterials are also assessed in order to test the effects of differentcombinations of matrix and stem cells.

Six rats are assigned to each treatment group and the defects are filledwith the designated matrix and cell combination. At four weeks, serum iscollected and rats are sacrificed. Serum is tested for immunologicreaction to the implants. Rat crania are collected for microradiographyand placed in 10% NBF.

Calvariae are processed for paraffin embedding and sectioning. Coronalhistological sections of the calvariae are stained with toluidine stainaccording to conventional techniques. Bone ingrowth into the defect andremnant of matrix carrier is assessed according to a 0 to 4 scale, withfour being the largest amount of ingrowth. Inflammation and fibrosis isalso assessed.

Treatment of bone lesions resulting from cancer metastases can beassessed according to an adaptation of the procedure of Bauerle et al.,2005, Int. J. Cancer 115:177-186. Briefly, site-specific osteolyticlesions are induced in nude rats by intra-arterial injection of humanbreast cancer cells into an anastomosing vessel between the femoral andthe iliac arteries. The metastases are then either treated withconventional anti-cancer therapies (e.g., chemotherapeutic,radiological, immunological, or other therapy) or surgically removed.Next, the lesions remaining from the cancer metastases are filled withdifferent matrix combinations as described above. After an appropriateperiod of time, as determined by radiologically monitoring the animals,the animals are sacrificed. Immunologic response against the matrix,inflammation, fibrosis, degree of bone ingrowth, and amount of matrixcarrier are assessed.

Additional references that describe models of bone disease that can beused or adapted to assess the efficacy of compositions comprisingplacental stem cells to treat bone defects include Mitsiades et al.,2003, Cancer Res. 63:6689-96; Chakkalakal et al., 2002, AlcoholAlcoholism 37:13-20; Chiba et al., 2001, J. Vet. Med. Sci. 63:603-8;Garrett et al., 1997, Bone 20:515-520; and Miyakawa et al., 2003,Biochem. Biophys. Res. Comm. 313:258-62.

6.12 Example 12: Production of Mineralized Collagen from Human PlacentaCollagen

A 4° C. human placental collagen (HPC) solution at ˜3 mg/ml was combinedwith a neutralizing buffer (200 mM Na₂HPO₄, pH 9.2) in an 85:15 ratio togive a final Na₂HPO₄ concentration of 30 mM and a pH of 7.2. Slight pHadjustments were accomplished with the addition of 1 N NaOH or HCl whilestirring. Once the pH was adjusted, stirring was stopped and thereaction was ramped at 1° C./min to 32° C. The reaction was isothermedfor 20-24 hours and the fibrillar collagen was isolated bycentrifugation. The collagen was resuspended 3× with phosphate bufferedsaline (PBS, 20 mM Na₂HPO₄, 130 mM NaCl, pH 7.4) and centrifuged toisolate collagen. The final washed fibrillar collagen was resuspended to10 mg/ml in PBS and stored at 4° C. until used. Fibrillation of HPCreconstitutes the soluble collagen as short fibrils and long fibers asshown in FIG. 22 a.

6.12.1 Mineralization of Collagen Fibrils

To mineralize the collagen, Ca(OH)₂ was dispersed at 199.9 mmol/L whilea 59.7 mM solution of H₃PO₄ was made. The Ca(OH)₂ and the H₃PO₄ werecombined together in a 2:1 ratio, respectively, and the pH was adjustedto 9 in a water jacket reaction vessel. This produces a 1.67 Ca/P ratio.The reaction was stirred vigorously while the temperature was held at40° C. and the pH was held at 9 by a circulating water bath and anautomatic titration unit, respectively. Fibrillar collagen in PBS wasslowly added to the reaction mixture and the pH was returned to 9. Thefinal mineral to collagen ration was 80:20. The reaction was stirredvigorously for 18 hours and the mineralized collagen (MC) was isolatedby centrifugation and washed 3 times with PBS. During the mineralizationreaction a Ca—P mineral formed along the fibers as shown in theelectromicrograph presented as FIG. 22b . The final reaction yield washigh (>80%), and the final mineral/collagen ratio of the material wasclose to the input mineral/collagen ratio as determined using TGA (FIG.23).

6.12.2 Crosslinking of Composite

The mineralized collagen (MC) was resuspended to approximately 2.5 mg/mlcollagen in PBS and placed in a water jacket reaction vessel. The pH wasadjusted to 9.5 and held constant throughout the reaction with anautomatic titration unit, while the temperature was held constant at 25°C. with a circulating water bath. Butane diol digycidyl ether (BDDE) wasadded to a final concentration of 50 mM. The reaction was stirredvigorously for 24 hours at which time the product was isolated bycentrifugation, washed once with PBS, and resuspended in PBS with 0.5Mglycine (pH 10) to quench any unreacted residual epoxide groups. Thereaction was stirred vigorously at 25° C. for 24 hours and then washed 3times with PBS. Centrifugation was used to isolate the crosslinkedmineralized collagen (CMC). The CMC formulations were characterized bylight and scanning electron microscopy, Thermo Gravimetric Analysis(TGA), Differential Scanning calorimetry (DSC) X-ray diffractometer(XRD), and Fourier Transform Infrared Spectroscopy (FTIR).

Crosslinking was confirmed by an increase in the denaturationtemperature of the collagen from ˜50 to ˜70° C. as determined by DSC.The crosslinked material had more mechanical integrity than thenon-crosslinked material and appeared more fibrous when examined bystereo microscopy and scanning electron microscopy (SEM). FTIR indicatedthe presence of a carbonated calcium phosphate mineral. XRD confirmedthat the mineral is a poorly crystallized hydroxyapatite.

6.13 Example 13: Growth of PDACS on a Mineralized Human PlacentalCollagen Matrix

This Example describes the results of experiments assessing the abilityof adherent placental stem cells to attach and grow on a mineralized HPCmatrix. In these experiments, CMCs produced as described above weresterilized with antibiotic and antimycotic reagents. Wet samples wereloaded into transwells for non-contact cytotoxicity studies usingplacental stem cells in a standard lactose dehydrogenase cytotoxicityassay (LDH) according to the manufacturer's instructions. LDH releasedinto the culture medium was correlated to cytotoxicity.

Next, CMC prepared as described above was used for PDAC adhesion andproliferation studies. Placental stem cells were seeded onto CMC asdescribed above. PDAC cell numbers were analyzed using a PicoGreen DNAassay at 1, 5 and 7 days (Molecular Probes; Eugene, Oreg.). PDACs showedsimilar LDH production when exposed to CMC as when exposed to tissueculture polystyrene (TCPS), indicating low cytotoxicity of CMCs. PDACsalso attached in greater numbers to CMC than to non-crosslinkedmineralized collagen at all seeding densities tested. Seven days afterseeding, this trend continued, with placental stem cells having thehighest cell numbers on CMC.

6.14 Example 14: Repair of Cranial Defects Using Placental Stem Cellsand Implantable Matrix

Tissue engineering using stem cells is emerging as a promisingalternative to tissue or organ transplantation. Novel stem cellsisolated from postpartum placenta (Placenta-Derived Adherent Cells,PDACs) have characteristics and phenotype of multi-potential stem cells.PDACs constitute an important and non-controversial source ofstem/progenitor cells that could be used as a therapeutic option for therepair of damaged or diseased tissue. In the present study, weinvestigated the osteogenic behavior of PDACs in vitro and in vivo.

Methods

In vitro study: Placental stem cells were obtained from the placenta byphysical disruption of tissue from different anatomical sites, seeded inbasal medium, and then induced with osteogenic differentiation medium(OS) as described above. The in vitro osteogenesis activity of PDACs wasevaluated by alkaline phosphatase (AP) activity and mineralization ofthe extracellular matrix was detected by Alizarin Red staining.Placental stem cell loading and viability on 3 dimensional scaffolds wasdetermined using a DNA assay and the CELLTITER GLO® Luminescent assayrespectively.

In vivo study: Placental stem cells were loaded on scaffolds (eitherVITOSS® Orthovita or HEALOS™ DePuy) and cultured for up to 1 hour invitro to form cell/scaffold constructs for implantation. For the ectopicmodel, placental stem cell-loaded VITOSS® constructs were implantedsubcutaneously into 40 athymic rats and collected 6 weeks afterimplantation. Explants were analyzed by immuno-histochemistry (IHC). Forthe bone defect model, bilateral cranial defects (3 mm×5 mm) werecreated in 96 male Hsd:RH-Foxn^(rmu) athymic rats (Charles River,Wilmington, Mass.), and used to compare the osteogenic/repair potentialof placental stem cells+HEALOS™, bone morphogenic protein-2(BMP-2)+HEALOS™ as a positive control, scaffold (HEALOS™) alone as anegative control, and empty defects (no treatment). Rats wereapproximately 6 weeks old at the time of the study, and sixteen ratswere assigned to each group. Explants for experimental conditions wereloaded with 500 μL of a stem cell suspension at 5×10⁶ cells permilliliter. Positive control comprised 5 μg BMP-2 per 25 mg carrier.Negative control comprised HEALOS with 500 μL cell culture medium.Explants were collected at 3 or 7 weeks after implantation, and analyzedwith microradiograph, mineralized tissue density (imagingsoftware-ImageJ 1.37v), Lunar PIXI x-ray densitometer, and histology.Histology was performed on excised bone tissue using hematoxylin &eosin, T-blue and vimentin stains.

Results

The in vitro osteogenic behavior of placental stem cells wasdemonstrated by the induction of AP activity and the cells' capacity toform Alizarin Red positive deposits. In vivo results: The placental stemcell+VITOSS® subcutaneous explants showed positive immunohistochemicalstaining for human osteocalcin, demonstrating the in vivo osteogenicpotential of the placental stem cells. In the cranial defect study, 3week placental stem cell+HEALOS™ explants presented considerable boneformation on histology and high density mineralization on x-ray andPIXI; these osteogenic activities were increased at 7 weeks afterimplantation. Representative histology slides, micro radiographs, andsemi-quantitative measurement of mineralization of the defect area aredepicted in FIGS. 24-26. These results demonstrate the ability ofplacental stem cells, in conjunction with a scaffold, to augment thebone repair process.

Conclusions: Adherent placental stem cells differentiate functionallyalong an osteogenic pathway given the appropriate stimuli in vitro, anddemonstrate significant enhancement of bone repair in vivo as comparedto cell-free conditions. Therefore, from these studies we conclude thatplacental stem cells can be used as a cellular therapeutic in bonetissue engineering applications with proper scaffolds.

EQUIVALENTS

The compositions and methods provided herein are not to be limited inscope by the specific embodiments described herein. Indeed, variousmodifications of the embodiments in addition to those described willbecome apparent to those skilled in the art from the foregoingdescription and accompanying figures. Such modifications are intended tofall within the scope of the appended claims.

Various publications, patents and patent applications are cited herein,the disclosures of which are incorporated by reference in theirentireties.

1-4. (canceled)
 5. A method for treating bone defects in a subject,comprising administering to a subject in need thereof an implantable orinjectable composition comprising a population of nonadherent stemcells, thereby treating the bone defect in the subject.
 6. The method ofclaim 5, wherein the bone defect is an osteolytic lesion associated witha cancer, a bone fracture, or a spine in need of fusion.
 7. The methodof claim 6, wherein the osteolytic lesion is associated with multiplemyeloma, bone cancer, or metastatic cancer.
 8. The method of claim 6,wherein the bone fracture is a non-union fracture.
 9. The method ofclaim 5, wherein the implantable composition is surgically implanted.10. The method of claim 5, wherein an injectable composition comprisinga population of nonadherent stem cells is administered to the subject.11. The method of claim 5, wherein the injectable composition issurgically administered to the region of the bone defect.
 12. The methodof claim 5, wherein the injectable composition is systemicallyadministered. 13-14. (canceled)
 15. A method for formulating aninjectable composition, comprising combining a population of stem cellswith injectable hyaluronic acid or collagen, wherein said stem cells areCD200+ or HLA-G+, or are CD34+ and CD44−.
 16. A method for treating bonedefects in a subject, comprising administering to a subject in needthereof an implantable or injectable composition comprising a populationof stem cells, wherein said stem cells are CD200+ or HLA-G+, or areCD34+ and CD44−, thereby treating the bone defect in the subject. 17.The method of claim 15, wherein the bone defect is an osteolytic lesionassociated with multiple myeloma, bone cancer, or metastatic cancer, abone fracture, or a spine in need of fusion.
 18. The method of claim 15,wherein an implantable composition comprising a population ofnonadherent stem cells is administered to the subject.
 19. The method ofclaim 17, wherein the implantable composition is surgically implanted.20. The method of claim 15, wherein an injectable composition comprisinga population of nonadherent stem cells is administered to the subject.21. The method of claim 19, wherein the injectable composition issystemically administered.